Compositions and methods for the treatment of disease associated with trp-p8 expression

ABSTRACT

Provided are small-molecule Trp-p8 modulators, including Trp-p8 agonists and Tip-p8 antagonists, and compositions comprising small-molecule Trp-p8 agonists as well as methods for identifying and characterizing novel small-molecule Trp-p8 modulators and methods for decreasing viability and/or inhibiting growth of Trp-p8 expressing cells, methods for activating Trp-p8-mediated cation influx, methods for stimulating apoptosis and/or necrosis, and related methods for the treatment of diseases, including cancers such as lung, breast, colon, and/or prostate cancers as well as other diseases, such as benign prostatic hyperplasia, that are associated with Trp-p8 expression.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. 119(e) to U.S.Provisional Patent Application No. 60/497,384 filed Aug. 22, 2003.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to the fields of cell biology,biochemistry, and organic chemistry. More specifically, the presentinvention provides small-molecule modulators of Trp-p8 activity, whichinclude Trp-p8 agonists and Trp-p8 antagonists, as well as compositionscomprising small-molecule Trp-p8 modulators. Also provided are methodsfor identifying and characterizing novel small-molecule Trp-p8modulators as well as methods for modulating Trp-p8-mediated cationinflux and/or apoptosis in a cell and related methods for the treatmentof diseases associated with Trp-p8 expression, activation, and/orsignaling. Exemplary diseases suitably treated by the compositions andmethods of the present invention include cancers, such as lung, breast,colon, and/or prostate cancers.

BACKGROUND OF THE INVENTION

Prostate carcinoma is the most common cancer diagnosed in men in theUnited States and has the second highest cancer death rate yielding onlyto lung adenocarcinoma. Parker et al., CA Cancer J. Clin. 46:5-27(1996). Although it is possible to effectively treat organ-confinedprostate cancer, there are very limited treatment options for metastaticdisease. Thus, it is of great importance to find novel ways to diagnoseearly stage disease and to closely monitor both progression andtreatment of the disease, as well as to develop new therapeuticapproaches. To achieve this, it is important to understand the molecularmechanisms of prostate cancer development and to identify newbiochemical markers for disease diagnosis and progression.

To date there are very few prostate-specific markers available. Thebest-known and well-characterized markers of proven prostate cancerdiagnostic value are the proteins prostatic acid phosphatase (PAP),prostate specific antigen (PSA), and prostate-specific membrane antigen(PSMA). Each of these proteins has also become the target for novelimmunotherapy approaches to the treatment of disease. Horoszewicz etal., Anticancer Res. 7:927-935 (1987); Barren et al., Prostate 30:65-68(1997); Murphy et al., Prostate 33:281-285 (1997); Murphy et al.,Prostate 26:164-168 (1995); Rochon et al., Prostate 25:219-223 (1995);Correale et al., J. Immunol. 161:3186-3194 (1998); and Murphy et al.,Prostate 38:73-78 (1999).

It has been reported that a cation channel protein, variously designatedTrp-p8 (transient receptor potential-p8), TRPM8, and CMR1 (cold andmenthol receptor 1), is preferentially expressed in prostate. Cloning ofthe full-length human trp-p8 cDNA revealed a transcript corresponding toan 1104 amino acid polypeptide sharing homology with the trp family ofcalcium channels. Clapham et al., Nature Reviews 2:387-396 (2001) andClapham et al., IUPHAR Compendium, TRP Channels (2002). Trp-p8 showsparticularly high homology with the human TRPC7 gene—a putative Ca²⁺channel protein of the trp family that is highly expressed in braintissue. Nagamine et al., Genomics 54:124-131 (1998). Trp-p8 also showssignificant homology to human melastatin, another Trp family-relatedprotein expressed in melanocytes and believed to be a tumor suppressorgene. Duncan et al., Cancer Res. 58:1515-1520 (1998); and Hunter et al.,Genomics 54:116-123 (1998). Perhaps of greatest interest is theobservation that the trp-p8 gene appears to be expressed in a largespectrum of nonprostatic in addition to prostatic neoplastic lesions.Tsavaler et al., Cancer Res. 61(9):3760-9 (2001).

The Trp superfamily comprises more than 20 related cation channelproteins that have been implicated in processes including sensoryphysiology to vasorelaxation and male fertility. Defects in Trp channelshave been associated with changes in growth control and tumorsuppression. While all Trp proteins are calcium channels, they varysignificantly in their selectivity and mode of activation. Members ofthe Trp superfamily share significant sequence homology and predictedstructural similarities, such as size of predicted transmembranesegments.

Trp-p8 is over-expressed in a range of cancers including prostate,breast, lung and colon, while within normal tissues, it is predominantlyexpressed in human prostate [Tsavaler et al., supra] and dorsal rootganglia (DRG, Dendreon, unpublished observation). Fuessel et al.reported that Trp-p8 is a highly prostate-specific and prostatecarcinoma-associated gene thus qualifying it as a potential target forspecific therapies. International J. of Oncology 23:221-228 (2003).Among other species, Trp-p8 orthologues are reportedly expressed in asubset of DRG and trigerminal ganglia (TG) neurons in rat [McKemy etal., Nature 416(6876):52-8 (2002)] and mouse [Peier et al., Cell108(5):705-15 (2002)] as well. Thus, Trp-p8 is a pantumor-expressedmarker with significant potential use in disease diagnosis andmonitoring of disease progression during treatment as well as a viabletarget for cancer therapy.

In two articles published concurrently, it was reported for the firsttime that Trp-p8 orthologues, in response to cold and certain coolingcompounds, initiate an influx of cations, such as calcium, from theextracellular space. McKemy et al., supra; and Peier et al., supra. Twoof the best known modulators of Trp-p8 activity are the Trp-p8 agonistsmenthol and Icilin. Menthol is effective in inducing calcium influx at^(˜)10-100 μM while Icilin is more potent with an effectiveconcentration range of 0.1-1 μM.

The higher temperature threshold reported for Trp-p8 activation by themost widely studied agonist, menthol(2-isopropyl-5-methyl-cyclohexanol), is about 30-32° C. in a variety ofcells (cold-sensitive neurons, Trp-p8 heterologously expressed inXenopus oocytes, HEK293 and CHO cells). McKemy et al., Nature, supra;Peier et al., Cell, supra; Nealen et al., J Neurophysiol. 90(1):515-520(2003); and Reid et al., J Physiol. 545(Pt 2):595-614 (2002).

Although certain agonist compounds have been shown to activate Trp-p8expressing cells up to 32° C., there has been no report disclosinggrowth manipulation in cells at physiological temperature (i.e. 37° C.),the temperature at which a compound must be active in order to be anefficacious in vivo therapeutic.

Association of Trp-p8 with prostate, lung, breast, and colon cancers andthe important role various ion channels play in vital cell functionssuggest that Trp-p8 channel may have a significant function in cancercell signaling and/or proliferation. Modulation of Trp-p8 activity,either by activating via an agonist or inhibiting via an antagonist, ata physiological temperature can be valuable as a therapeutic tomanipulate the Trp-p8 expressing cells in a specific manner.

Accordingly, there remains a need in the art for small-moleculemodulators of Trp-p8 activity, compositions comprising one or moresmall-molecule Trp-p8 modulators, and methods for the identification anduse of small-molecules for modulating the activity of Trp-p8 in a celland for the treatment of disease associated with the aberrant expressionof Trp-p8.

SUMMARY OF THE INVENTION

The present invention fulfills these and other related needs byproviding small molecule modulators of Trp-p8 activity, including Trp-p8agonists and Trp-p8 antagonists, as well as compositions comprising suchTrp-p8 modulators, and methods for identifying and using Trp-p8modulators. Within certain embodiments, compounds of the presentinvention bind to and activate Trp-p8 and/or stimulate cation influx,including but not limited to calcium influx, in a cell wherein cationinflux is correlative of Trp-p8 modulator induced toxicity. Thus, withinthese and other embodiments, Trp-p8 agonists of the present inventionare effective in inhibiting growth of and/or inducing apoptosis and/ornecrosis in a cell expressing Trp-p8. Within alternative embodiments areprovided Trp-p8 antagonists that are effective in reducing the basalactivity of Trp-p8 in a cell thereby reducing the viability of Trp-p8expressing cells. Advantageously, therefore, agonists and antagonists ofthe present invention can be used to treat diseases including, but notlimited to, cancers of the breast, lung, colon, and/or prostate, thatare associated with Trp-p8 expression.

One or more Trp-p8 modulator can be formulated in compositions,including pharmaceutical compositions, comprising one or morepharmaceutically acceptable carrier or excipient and/or one or moreadditional therapeutic compound. Such compositions will find utility inmethods for the treatment of one or more disease associated with Trp-p8expression.

Thus, in one embodiment, the present invention provides the followingTrp-p8 modulators and derivatives thereof:

A compound of Formula I

wherein

R¹ is selected from the group consisting of H, OH, CH₃, CH₃—CH—CH₃(isopropyl), and CH₃—C═CH₂ (isopropenyl);

R² is selected from the group consisting of H;

R³ is selected from the group consisting of O, OH, acetate, lactate,carboxamide, butanamide, sulphanamide, and propanetriol; and

R⁴ is selected from the group consisting of CH₃—CH—CH₃ (isopropyl),isopropane-2-ol, and CH₃—C═CH₂ (isopropenyl).

Exemplary compounds of Formula I include the following:

These exemplary Formula I Trp-p8 modulators have the following trade andchemical names: Menthol (2-isopropyl-5-methyl-cyclohexanol)(Sigma-Aldrich, Inc.; St. Louis, Mo.); Frescolat ML (Harris & Ford, LLC;Indianapolis, Ind.; Menthyl lactate); L-Menthyl Acetate (MillenniumChemicals; Olympia Fields, Ill.;Cyclohexanol-5-methyl-2-(1-methylethyl)-acetate-[1R-(1alpha,2beta,5alpha)]-); Cooling Agent 10 (Takasago International Corp.;Rockleigh, N.J.; (1)-Menthoxypropane-1,2-diol); Coolact P® (TakasagoInternational Corp.; (−)-Isopulegol); and Coolact 38D® (TakasagoInternational Corp.).

In another embodiment, the present invention further provides thefollowing small-molecule Trp-p8 modulators and derivatives thereof:

A compound of Formula II

wherein

R⁵ is selected from the group consisting of H, OH, CH₃, CH₃—CH—CH₃(isopropyl), and CH₃—C═CH₂ (isopropenyl);

R⁶ is selected from the group consisting of N;

R⁷ is selected from the group consisting of O and N;

R⁸ is selected from the group consisting of NH, O, and S; and

R⁹ is selected from the group consisting of NO₂.

Compounds of Formula II are exemplified herein by Icilin(1-(2-hydroxyphenyl)-4-(3-nitrophenyl)-1,2,3,6-tetrahydropyrimidine-2-oneaka3,4-dihydro-3-(2-hydroxyphenyl)-6-(3-nitrophenyl)-(1H)-pyrimidin-2-one).

In another embodiment, the present invention further provides thefollowing acyclic carboxamide Trp-p8 agonists and derivatives thereof aspresented in U.S. Pat. No. 4,153,679, incorporated herein by reference:

A compound of Formula III

wherein

R¹⁰ is selected from the group consisting of H and a C₁-C₅ alkylincluding, but not limited to, CH₃, C₂H₅, CH₃—CH—CH₃ (isopropyl) andCH₃—C═CH₂ (isopropenyl);

R¹¹ is selected from the group consisting of OH, carboxamide,butanamide, propanetriol, and CONR′R″, wherein R′ is selected from thegroup consisting of H, CH₃,

C₂H₅, C₄H₈ (cyclobutyl), and C₄H₈O, and wherein R″ is selected from thegroup consisting of C₂H₅OOCH₂, C₂H₅, CH₃—CH—CH₃ (isopropyl),HOCH₂C(CH₃)₂, HOCH₂CH₂, C₄H₉ (tertbutyl), and C₄H₉ (secbutyl);

R¹² is selected from the group consisting of H and a C₁-C₅ alkylincluding, but not limited to, CH₃, CH₃—CH—CH₃ (isopropyl), CH₃—C═CH₂(isopropenyl), C₄H₉ (secbutyl), C₄H₉ (isobutyl), C₄H₉ (n-butyl), andC₅H₁₁ (isohexyl); and

R¹³ is selected from the group consisting of H and a C₁-C₅ alkylincluding, but not limited to, CH₃, C₂H₅, CH₃—CH—CH₃ (isopropyl),CH₃—C═CH₂ (isopropenyl), C₄H₉ (secbutyl), and C₄H₉ (isobutyl).

Compounds of Formula III are exemplified herein by WS-23(2-Isopropyl-N,2,3-trimethylbutyramide aka N,2,3-trimethyl-2-isopropylbutamide).

In another embodiment, the present invention further provides thefollowing 3-substituted-p-menthane Trp-p8 modulators and derivativesthereof exemplified by those presented in U.S. Pat. No. 4,150,052,incorporated herein by reference:

A compound of Formula IV

wherein

R¹⁴ is selected from the group consisting of H and an aliphatic groupcontaining up to 25 carbon atoms;

R¹⁵ is selected from the group consisting of H, OH and an aliphaticgroup containing up to 25 carbon atoms, with the proviso that when R¹⁵is H, R¹⁴ may also be an aryl group of up to 10 carbon atoms andselected from the group consisting of substituted phenyl, phenalkyl,substituted phenalkyl, naphthyl, substituted naphthyl, and pyridyl; and

R¹⁴ and R¹⁵, when taken together with the nitrogen atom to which theyare attached, may form a cyclic or heterocyclic group of up to 25 carbonatoms, e.g., a piperidino or a morpholino group. Exemplary such cyclicalgroups may be selected from the group consisting of3-phenyl-piperidin-1-yl, 3-phenyl-pyrrolidin-1-yl,6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-yl, and4-pyrimidin-2-yl-piperazin-1-yl.

Typical values for R¹⁴ and R¹⁵ when aliphatic are methyl, ethyl, propyl,butyl, isobutyl, n-decyl, cyclopropyl, cyclohexyl, cyclopentyl,cycloheptylmethyl, 2-hydroxyethyl, 3-hydroxy-n-propyl,6-hydroxy-n-hexyl, 2-aminoethyl, 2-acetoxyethyl, 2-ethylcarboxyethyl,4-hydroxybut-2-ynyl, and carboxymethyl.

When R¹⁴ is aryl, typical values are benzyl, naphthyl, 4-methoxyphenyl,2-methoxy-4-methoxyphenyl, 3-methoxy-5-methoxyphenyl,4-methyl-5-chlorophenyl, 4-hydroxyphenyl, 4-methylphenyl,3-methyl-4-methylphenyl, 3-hydroxy-4-methylphenyl, 4-fluorophenyl,4-chlorophenyl, 4-bromophenyl, 4-ethylphenyl, 2-fluoro-4-fluorophenyl,4-nitrophenyl, 2-hydroxynaphthyl, pyridyl,[1-carbamoyl-2-(1H-indol-3-yl)-ethyl,1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl,1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl,1-carbamoyl-2-(4-hydroxyphenyl)ethyl, 1-carbamoyl-2-phenylethyl,1-hydroxymethyl-2-(1H-indol-3-yl)ethyl,1-hydroxymethyl-2-(4-chlorophenyl)ethyl,1-hydroxymethyl-2-(4-hydroxyphenyl)ethyl,1-hydroxymethyl-2-hydroxy-2-phenylethyl, 1-hydroxymethyl-2-phenyl ethyl,1-methoxymethyl-2-phenylethyl, 1-methyl-2-(4-chlorophenyl)-ethyl,1-methyl-2-(5-fluoro-1H-indol-3-yl)-ethyl,1-methyl-2-hydroxy-2-phenylethyl, 1-oxo-2-phenylethyl,1-phenyl-cyclopentylmethyl, 2-(1-cyclopentyl-pyrrolidin-3-yl)-ethyl,2-(1H-indol-3-yl)ethyl, dimethoxyphenyl)ethyl,2-(2,4-dichlorophenyl)ethyl, 2-(2,4-dimethylphenyl)ethyl,2-(2,5-dimethoxyphenyl)ethyl, 2-(2,5-dimethylphenyl)-ethyl,2-(2,6-dimethylphenyl)ethyl, 2-(2-chloro-6-fluorophenyl)ethyl,2-(2-chlorophenyl)ethyl, 2-(2-fluorophenyl)ethyl, 2-(2-furyl)ethyl,2-(2-methoxy-5-bromophenyl)ethyl, 2-(2-methoxyphenyl)-ethyl,2-(2-methylphenyl)ethyl, 2-(3,4-dichlorophenyl)ethyl,2-(3,4-dimethoxyphenyl)ethyl, 2-(3,5-dimethoxyphenyl)ethyl,2-(3-bromo-4-methoxyphenyl)ethyl, 2-(3-chlorophenyl)ethyl,2-(3-ethoxyphenyl)ethyl, 2-(3-fluorophenyl)ethyl,2-(3-hydroxy-4-methoxyphenyl)-ethyl, 2-(3-hydroxyphenyl)ethyl,2-(3-methoxy-4-ethoxyphenyl)ethyl, 2-(3-methoxy-4-hydroxyphenyl)ethyl,2-(3-methoxyphenyl)ethyl, 2-(3-methylphenyl)ethyl,2-(3-trifluoromethylphenyl)ethyl, 2-(4-bromophenyl)-ethyl,2-(4-chlorophenyl)ethyl, 2-(4-ethylphenyl)ethyl,2-(4-fluorophenyl)ethyl, 2-(4-hydroxyphenyl)ethyl,2-(4-methoxy-phenyl)-2-oxo-ethyl, 2-(4-methoxyphenyl)ethyl,2-(4-methylphenyl)ethyl, 2-(4-methylphenyl)ethyl,2-(4-methylsulfanylphenyl)ethyl, 2-(4-nitrophenyl)ethyl,2-(4-sulfamoyl-phenyl)-ethyl, 2-(5-methoxy-1H-indol-3-yl)-ethyl,2-(6-fluoro-1H-indol-3-yl)-ethyl, 2-(6-methoxy-1H-indol-3-yl)-ethyl,2-(7-methyl-1H-indol-3-yl)-ethyl, 2-(N,N-dipropylamino)ethyl,2-(pyridin-2-yl)-ethyl, 2-(pyridin-3-yl)-ethyl, 2-(pyridin-4-yl)-ethyl,2,2-diphenylethyl, 2,3-difluorobenzyl, 2,3-dimethoxyphenyl,2,4-dichlorophenyl, 2,4-difluorophenyl, 2,4-dimethoxyphenyl,2,4-dimethylphenyl, 2-bromo-4-methylphenyl, 2-chloro-4-cyanophenyl,2-chloro-4-fluorophenyl, 2-chloro-4-iodophenyl, 2-chloro-4-nitrophenyl,2-chloro-5-nitrophenyl, 2-chlorophenyl, 2-cyclohex-1-enyl-ethyl,2-fluoro-4-chlorophenyl, 2-fluoro-5-nitrophenyl,2-hydroxy-2-(3-hydroxyphenyl)ethyl, 2-hydroxy-2-(4-hydroxyphenyl)ethyl,2-hydroxy-2-phenylethyl, 2-iodophenyl,2-methyl-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl, 2-methyl-2-phenylethyl,2-methyl-4-bromophenyl, 2-methyl-5-nitrophenyl, 2-methylphenyl,2-nitro-4-fluorophenyl, 2-nitrophenyl,2-oxo-2,3-dihydro-1H-benzoimidazol-5-yl, 2-oxo-2-phenylethyl,2-phenyl-1H-benzoimidazol-5-yl, 2-phenyl-2-(4-chlorophenyl)ethyl,2-phenyl-2-(4-fluorophenyl)-ethyl, 2-phenyl-2-(4-methoxyphenyl)ethyl,2-phenylethenyl, 2-phenylethyl, 2-pyridin-2-yl-benzooxazol-5-yl,2-pyridin-3-yl-1H-benzoimidazol-5-yl, 2-thiophen-2-yl-ethyl),2-trifluoromethyl-1H-benzoimidazol-5-yl, 3,4,5-trifluorophenyl,3,4,5-trimethoxyphenyl, 3,4-cyclopentanephenyl, 3,4-dichlorophenyl,3,4-dimethylphenyl, 3,5-dimethoxyphenyl, 3-acetamidophenyl,3-bromo-4-methylphenyl, 3-carboxamidophenyl, 3-chloro-4-methoxyphenyl,3-chloro-4-methylphenyl, 3-chloro-4-morpholin-4-yl-phenyl,3-hydroxymethylphenyl, 3-nitrophenyl, 3-oxo-indan-5-yl, 3-phenylpropyl,3-yl-acetophenone, 4-(1,1-dioxo-1l6-thiomorpholin-4-ylmethyl)-phenyl,4-(1-hydroxyethyl)phenyl, 4-(2-hydroxyethyl)phenyl,4-(4-ethyl-piperazin-1-yl)-phenyl,4-(4-methyl-1H-benzoimidazol-2-yl)-phenyl,4-(4-methyl-piperazin-1-ylmethyl)-phenyl,4-(morpholine-4-sulfonyl)-phenyl,4-[1,3-dioxo-2-(2-trifluoromethyl-phenyl)-2,3-dihydro-1H-isoindol-5-yloxy]-phenyl,4-[2-(2-methoxy-phenyl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yloxy]-phenyl,4-{2-[2-(3,4-dimethoxy-phenyl)-ethyl]-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yloxy}-phenyl,4-acetylphenyl, 4-azepan-1-ylmethyl-phenyl, 4-benzooxazol-2-yl-phenyl,4-Biphenyl, 4-bromophenyl, 4-carboxamidophenyl, 4-chlorophenyl,4-cyanophenyl, 4-ethoxyphenyl, 4-ethylphenyl, 4-fluorophenyl,4-hydroxymethylphenyl, 4-methoxyphenyl, 4-methyl-2-oxo-2H-chromen-7-yl,4-methylcyclohexyl, 4-methylphenyl, 4-methylsulfanylphenyl,4-nitrobenzyl, 4-pyrrolidin-1-ylmethyl-phenyl, 4-trifluoromethylphenyl,benzo[1,3]dioxol-5-yl, benzoylamino, benzyloxy, bicyclo[2.2.1]hept-2-yl,C-1H-indazol-5-yl, cycloheptyl, indan-2-yl,N-(2-diethylamino-ethyl)-benzamide-4-yl, N′-quinoxalin-2-yl-amino, andphenylcyclopropyl.

Trp-p8 modulators of Formula IV are exemplified herein by the Trp-p8agonists WS-3 (Millennium Chemicals; N-Ethyl-p-menthane-3-carboxamideaka cyclohexanecarboxamide, N-ethyl-5-methyl-2(1-methylethyl)) and byWS-12 (N-(4-methoxyphenyl)-p-menthan-3-carboxamide akacyclohexanecarboxamide, N-(4-methoxyphenyl)-5-methyl-2(1-methylethyl)),and the compounds presented in Table 1.

In a further embodiment, the present invention provides the followingTrp-p8 modulators and derivatives thereof that comprise at least oneketal moiety, including, but not limited to the Trp-p8 agonists1-menthone glycerol ketal and 3,3,5-trimethylcyclohexaone glycerol ketalpresented in U.S. Pat. No. 5,266,592, incorporated herein by reference:

A compound comprising at least one ketal of Formula V

wherein

R¹⁶ is selected from the group consisting of a C₂-C₆ alkylene grouphaving at least one, but not more than three, hydroxyl group(s),preferably one hydroxyl group; and

either R¹⁷ and R¹⁸, independently of one another, represent C₁-C₁₀-alkylwhich is optionally substituted by 1 to 3 groups selected from the groupconsisting of hydroxyl, amino, thio, and halogen (e.g., fluorine,chlorine, bromine, or iodine), C₅-C₇-cycloalkyl, preferably cyclohexyl,C₆-C₁₂-aryl, preferably phenyl, with the proviso that the total of the Catoms of R¹⁷ and R¹⁸ is not less than 3; or

R¹⁷ and R¹⁸ together represent an alkylene group that, together with thecarbon atom that carries the groups R¹⁷ and R¹⁸, forms a 5-7-memberedring, it being possible for this alkylene group, in turn, to besubstituted by C₁-C₆-alkyl groups.

Preferred groups R¹⁷ and R¹⁸ comprise methyl, isopropyl, and tert-butyl.

Trp-p8 modulators comprising a ketal of Formula V include the followingcompounds wherein R¹⁶ is as defined above:

Compounds comprising a ketal of Formula V are exemplified herein by theTrp-p8 agonist Frescolat MGA (Harris & Ford, LLC; Menthone GlycerinAcetal),

In yet a further embodiment, the present invention provides Formula VIsmall-molecule Trp-p8 agonists and derivatives thereof exemplifiedherein by L-Carvone (Millennium Chemicals;(R)-5-Isopropenyl-2-methyl-2-cyclohexenone p-Mentha-6,8-dien-2-one),

Other embodiments of the present invention provide Trp-p8 modulators ofFormula VII. A compound of Formula VII

wherein

R¹⁷ is selected from the group consisting of 2-pyridyl,2-nitro-4-trifluoromethylphenyl, 2-nitro-4-chlorophenyl,2-methoxyphenyl, 2-chlorophenyl, phenyl, 2-methyl-quinolin-3-yl,4-methoxyphenyl, 4-fluorophenyl,3-azepan1-yl-5-(4-trifluoromethoxy)phenylamino[1,3,5]triazyl,cyclohexyl, diphenylmethyl, 2-phenylethyl, 4-hydroxy-cyclohexyl,cycloheptyl, cyclopentyl, C-benzo[1,3]dioxol-5-yl-methyl, 2-pyridyl, and4-chlorobenzyl;

R¹⁸ is selected from the group consisting of1-benzyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl, 3-benzylamino-2-nitrophenyl,5-nitro-quinolin-8-yl,1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol,1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,benzyl-2-methyl-quinazolin-4-yl,3-methyl-5-morpholin-4-yl-2-nitro-phenyl,2-nitro-5-piperazin-1-yl-ethanol,1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol,4-(2,5-dimethyl-pyrrol-1-yl)-2-nitro-phenyl,2-nitro-3-trifluoromethanesulfonyl-phenyl,1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,2-(2-Fluoro-phenoxymethyl)-2-cyano oxazolyl, adamantly,5-(benzo[1,3]dioxol-5-ylamino)-10b,10c-dihydro-anthra[1,9-cd]isoxazol-6-one-yl,2-methyl-thiazolo[3,2-b][1,2,4]triazol-6-ol 4-methylphenyl methyl,3-benzyl-3H-quinazolin-4-one-2-yl, cyclopentyl, tetrahydronapthyl,cyclooctyl, cyclohexyl,C-[3-(4-chloro-phenyl)-2,5-dimethyl-pyrazolo[1,5-a]pyrimidin-7-yl]-methyl,C-(2-benzyl-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-methyl,and 1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol;

R¹⁹ and R²⁰ are each independently selected from the group consisting ofH and O; and

R²¹ is selected from the group consisting of 4-methylphenyl,2-chloro-4-fluorophenyl, and 4-chlorophenyl.

In other embodiments, the present invention provides Formula VIIIsmall-molecule Trp-p8 modulators. A compound of Formula VIII

wherein

R²² is a linker moiety, which may be selected from the group consistingof oxyacetamide, urea, carbamate, thiourea, sulfonamide, amine, amide.Formula VIII antagonists are represented by the following sub-formulae(Formula VIII-A, Formula VIII-B, Formula VIII-C, Formula VIII-D, FormulaVIII-E, Formula VIII-F, and Formula VIII-G):

Irrespective of which of the seven R²² linker moieties is employed, R²³is selected from the group consisting of H, tetrahydro isoquinolinyl,tetrahydro quinolinyl, 3-methyl indolinyl, indolinyl, 2-(N-methyl,N-phenylethyl) amino ethyl, 3-methyl indolinyl, 1-phenyl ethyl, 2-chlorobenzyl, 2-methoxybenzyl, 2-methoxyphenyl, 2-cyclohex-1-enyl ethyl,(1-phenyl-cyclophenyl)-methyl, 2-(tetrahydroquinolinyl)-ethyl,1-propyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, cycloheptyl,3-cyclohexylsulfanylpropyl, 2-cyclohex-1-enyl ethyl, 2-(N-isopropyl,N-phenylethyl)amino ethyl,1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, 2-cyclopentylethyl,2-phenylcyclopropyl, 1-phenoxyethyl, 4-butyloxyphenyl,(2-nitrophenoxy)methyl,4,7,7-trimethyl-2-oxa-bicyclo[2.2.1]heptan-3-one,C-(1-phenyl-5-propyl-1H-pyrazol-4-yl)-methyl, benzyl, 2-chlorobenzyl,1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-methoxy-phenyl]-2,3,4,9-tetrahydro-1H-b-carboline,C-[3-(4-butoxy-phenyl)-1H-pyrazol-4-yl]-methyl,4-(azepane-1-sulfonyl)-phenyl, and5-(7-chloro-quinolin-4-ylsulfanyl)-[1,3,4]thiadiazol-2-yl;

R²⁴ is selected from the group consisting of H, tetrahydroisoquinolinyl, tetrahydro quinolinyl, 3-methyl indolinyl, indolinyl,3-methyl indolinyl, 1-propyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine,1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, and1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-methoxy-phenyl]-2,3,4,9-tetrahydro-1H-b-carboline;and

R²⁵ is selected from the group consisting of H.

Other aspects of the present invention provide compositions, includingpharmaceutical compositions, comprising one or more small-moleculeTrp-p8 modulators of Formula I, Formula II, Formula III, Formula IV,Formula V, Formula VI, Formula VII, and Formula VIII in combination witha pharmaceutically acceptable excipient, carrier and/or diluent.Exemplified herein within the Examples are specific Trp-p8 agonists ofFormula I, Formula II, Formula III, Formula IV, Formula V, and FormulaVI and Trp-p8 antagonists of Formula VII and Formula VIII. Also providedare Trp-p8 antagonists of Formula I, Formula II, Formula III, FormulaIV, Formula V, and Formula VI and Trp-p8 agonists of Formula VII andFormula VIII.

Within still further aspects, compositions of the present inventioncomprise one or more compound of Formula I, Formula II, Formula III,Formula IV, Formula V, Formula VI, Formula VII, and/or Formula VIIIformulated together with one or more cancer therapeutic agent.Alternatively, compositions of the present invention comprise a compoundof Formula I, Formula II, Formula III, Formula IV, Formula V, FormulaVI, Formula VII, and/or Formula VIII independently formulated with oneor more cancer therapeutic agent. That is, one or more compound ofFormula I, Formula II, Formula III, Formula IV, Formula V, Formula VI,Formula VII, and/or Formula VIII and the cancer therapeutic agent areseparately formulated.

Suitable cancer therapeutic agents include, but are not limited to,antimitotic agents including, but not limited to, paclitaxel,vincristine, and etoposide; alkylating agents including, but not limitedto, mechlorethamine, cyclophosphamide, and carmustine; antimetabolitesincluding, but not limited to, methotrexate, gemcitabine, lometrexol,5-fluorouracil, and 6-mercaptopurine; cytotoxic antibiotics including,but not limited to, doxorubicin, daunorubicin, bleomycin, mitomycin C,and streptozocin; platinum agents including, but not limited to,cisplatin and carboplatin; hormonal agents including, but not limitedto, anti-estrogens such as tamoxifen and diethylstilbestrol as well asanti-androgens such as flutamide; antiangiogenesis agents; and farnesyltransferase inhibitors.

In certain aspects, compounds of Formula I, Formula II, Formula III,Formula IV, Formula V, Formula VI, Formula VII, and/or Formula VIII areadministered in combination with cancer therapeutic agents that arethemselves ineffective for modulating Trp-p8 activity in a cellexpressing Trp-p8. Surprisingly, these types of combination therapyresult in enhanced efficacy relative to the use of a single compound ofFormula I, Formula II, Formula III, Formula IV, Formula V, Formula VI,Formula VII, and/or Formula VIII alone.

In other aspects, compounds of Formula I, Formula II, Formula III,Formula IV, Formula V, Formula VI, Formula VII, and/or Formula VIII areadministered in combination with one or more additional Trp-p8 modulatorincluding, but not limited to, a compound of Formula I, Formula II,Formula III, Formula IV, Formula V, Formula VI, Formula VII, and/orFormula VIII.

Within certain of these embodiments are provided small-moleculeantagonists of the small-molecule Trp-p8 agonists presented herein.Thus, within certain embodiments are provided small-molecule Trp-p8antagonists of Formula I, Formula II, Formula III, Formula IV, FormulaV, Formula VI, Formula VII, and/or Formula VIII, and derivativesthereof, of one or more Formula I, Formula II, Formula III, Formula IV,Formula V, Formula VI, Formula VII, and/or Formula VIII Trp-p8 agonist.

Further embodiments of the present invention provide methods fordecreasing cell viability and/or inhibiting cell growth, methods forstimulating cation influx, and methods for inducing apoptosis and/ornecrosis in a cell expressing Trp-p8. Exemplary such methods comprisethe step of contacting a cell with a compound of Formula I, Formula II,Formula III, Formula IV, Formula V, Formula VI, Formula VII, and/orFormula VIII in a concentration and for a time required to decrease cellviability and/or inhibit cell growth, to raise intracellular calcium,and/or to induce apoptosis and/or necrosis of the cell.

In still further embodiments, the present invention provides methods fortreating a disease in a mammal, most typically a human, by administeringone or more compound and/or composition of the present invention. Incertain aspects, the methods include the administration of a compositioncomprising a combination of a compound of Formula I, Formula II, FormulaIII, Formula IV, Formula V, Formula VI, Formula VII, and/or Formula VIEwith one or more cancer therapeutic agent delivered in a simultaneousmanner, such as in a single formulation. In certain other aspects, themethods of the present invention include combination therapy wherein thecompound of Formula I, Formula II, Formula III, Formula IV, Formula V,Formula VI, Formula VII, and/or Formula VIII is administered first inone formulation, followed by the cancer therapeutic agent in a separateformulation. The methods also include a cancer therapeutic agent beingdelivered first in one formulation, followed by a compound of Formula I,Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII,and/or Formula VIII in a separate formulation.

Therapeutic methods of the present invention are particularly effectivein the treatment of cancers associated with the expression of Trp-p8including, but not limited to, certain colon, lung, breast, and prostatecancers.

The above-mentioned and additional features of the present invention andthe manner of obtaining them will become apparent, and the inventionwill be best understood by reference to the following more detaileddescription, read in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph depicting an exemplary ATP viability assay. Trp-p8agonists were tested at 10 μM and agonist-specific killing of Trp-p8expressing CHO cells (CHO/Trp-p8) measured at 37° C.

FIGS. 2A-2C are graphs depicting Trp-p8 agonist-induced increases inintracellular calcium as determined by a calcium flux assay performed at37° C. FIG. 2A is a positive control demonstrating that CHO andCHO/Trp-p8 cells respond similarly to 1 μM lonomycin at 37° C. in thecalcium flux assay. FIG. 2B is a negative control demonstrating thatparental CHO cells that do not express endogenous or exogenous Trp-p8 donot respond to Trp-p8 agonists even at a concentration of 10 μM. FIG. 2Cdemonstrates that the Trp-p8 agonist, designated herein as compound1603, induced a specific, concentration-dependent response in CHO/Trp-p8cells at 37° C.

FIG. 3 are plots of flow cytometry data demonstrating that a Trp-p8agonist is capable of specifically inducing apoptosis in Trp-p8expressing CHO cells at 37° C.

FIG. 4 is a graph depicting exemplary results from a primary screen forTrp-p8 antagonists using the ATP viability assay, described herein, withCHO/Trp-p8 cells at 37° C. CHO/Trp-p8 cells were exposed to compounds,at different concentrations, in 1% DMSO or 1% DMSO in combination with atoxic concentration of the Trp-p8 agonist 1607. The viability of cellswas measured after 24-26 hours at 37° C. using the ATP assay. Thecompounds D-2258 and D-2212 protected the cells from the toxic effect ofthe Trp-p8 agonist D-1607 and, consequently, are classified as Trp-p8antagonists. D-2250 had no protective effect and is shown here for thepurpose of illustration of the assay.

FIG. 5 is a graph depicting the screening and characterization of Trp-p8antagonists by the calcium flux assay performed at 37° C. CHO/Trp-p8cells were loaded with the calcium indicator dye, Fura-2, and theincrease in intracellular calcium in response to compounds wasdetermined by the increase in fluorescence. Fura-2 dye loaded CHO/Trp-p8cells were exposed to 1% DMSO or D-2258, at different concentrations, in1% DMSO at 37° C. Three minutes later, D-1607 was added to the c ells.When c ells w ere exposed to effective concentrations of the antagonist,D-2258, their ability to respond to the agonist D-1607 was significantlyreduced or eliminated altogether.

SEQ ID NO: 1 is the nucleotide sequence of a human Trp-p8 cDNA (GenBankAccession No. AY090109).

SEQ ID NO: 2 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO: 1 (GenBank Accession No. NP_(—)076985).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the discovery that certainsmall-molecule agonists of Trp-p8 activity are capable of inhibiting thegrowth of and/or inducing apoptosis and/or necrosis in cells thatexpress Trp-p8. Without wishing to be limited to any specific mode ofaction, it is believed that Trp-p8 agonist-mediated activation of theTrp-p8 receptor substantially increases cation influx, which iscorrelative of cellular toxicity. I t is further believed that Trp-p8antagonists can inhibit the basal level and/or native ligand-inducedactivity of endogenous Trp-p8 activation which, consequently, leads t oreduced growth or death of cells expressing this cation channel protein.

Thus, the present invention provides small-molecule Trp-p8 modulators,including agonists and antagonists of Trp-p8 activity, as well ascompositions, including pharmaceutical compositions, comprising one ormore small-molecule Trp-p8 modulator in combination with one or morepharmaceutically acceptable carrier and/or excipient. The presentinvention also provides combination compositions comprising one or moreTrp-p8 modulator and one or more additional therapeutic compound suchas, for example, a cancer therapeutic agent. Trp-p8 modulators andcompositions comprising Trp-p8 modulators will find utility in methodsfor activating Trp-p8-mediated cation influx in a cell, methods forinducing apoptosis and/or necrosis in a cell, as well as methods for thetreatment of diseases associated with Trp-p8 expression including, butnot limited to, cancers, such as breast, colon, lung, and prostatecancers.

DEFINITIONS

The term “Trp-p8 modulators” refers collectively to small-moleculeagonists and antagonists that bind to and either increase or decrease,respectively, the activity of Trp-p8 in a cell. Trp-p8 agonists includecompounds of Formula I, Formula II, Formula III, Formula IV, Formula V,Formula VI, Formula VII, and Formula VIII and are exemplified herein byvarious compounds of Formulas I-VI, and chemical derivatives thereof.Trp-p8 antagonists include compounds of Formula I, Formula II, FormulaIII, Formula IV, Formula V, Formula VI, Formula VII, and Formula VIIIand are exemplified herein by various compounds of Formulas VII-VIII,and chemical derivatives thereof. Additional Trp-p8 agonists orantagonists of Formula I, Formula II, Formula III, Formula IV, FormulaV, Formula VI, Formula VII, and Formula VIII, not specificallyexemplified herein, may be readily synthesized and characterized by askilled artisan by employing the methodology expressly provided hereinand/or as is readily available in the art.

The phrase “activate Trp-p8” means agonist-mediated activation of Trp-p8expressed on the surface of a cell. For example, within certainembodiments, agonists of the present invention, when contacted with acell and/or administered in vivo to a mammalian subject, activate Trp-p8thereby facilitating the influx of cations, such as calcium ions, tosuch an intracellular level and/or for such a duration that issufficient to cause toxicity to the cell as evidenced by a diminution incell growth and/or an onset of necrotic and/or apoptotic cell death.

The term “aliphatic” is intended to include any straight-chained,branched-chained, or cyclic group free of aromatic unsaturation, andthus embraces alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl,hydroxyalkyl, acyloxyalkyl, alkoxy, alkoxyalkyl, aminoalkyl,acylaminoalkyl, carboxyalkyl, and similar combinations.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon group, or combination thereof, which may be fully saturated,mono or polyunsaturated and can include di and multivalent groups,having the number of carbon atoms designated (i.e. C₁-C₁₀ means one toten carbons). Examples of saturated hydrocarbon groups include groupssuch as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)ethyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups includevinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers.

The term “alkenyl” denotes branched or unbranched hydrocarbon chainscontaining one or more carbon-carbon double bonds.

The term “alkynyl” refers to branched or unbranched hydrocarbon chainscontaining one or more carbon-carbon triple bonds.

The term “alkylene” by itself or as part of another substituent means adivalent group derived from an alkane, as exemplified by —CH₂CH₂CH₂CH₂—.Typically, an alkylene group will have from 1 to 24 carbon atoms, withthose groups having 10 or fewer carbon atoms being preferred in thepresent invention. A “lower alkyl” or “lower alkylene” is a shorterchain alkyl or alkylene group, generally having eight or fewer carbonatoms.

The term “cycloalkylene” by itself or as part of another substituentmeans a divalent group derived from a cycloalkane, as exemplified bycyclohexylene. Typically, a cycloalkylene group will have from 5-8carbon atoms, with those groups having 6 carbon atoms being preferred inthe present invention.

The term “alkenylene” by itself or as part of another substituent meansa divalent group derived from an alkenyl, as exemplified by—CH═CHCH₂CH₂—. Typically, alkenylene groups will have from 2 to 24carbon atoms, with those groups having 10 or fewer carbon atoms beingpreferred in the present invention.

The terms “alkoxy,” “alkylamino” and “alkylthio” refer to those groupshaving an alkyl group attached to the remainder of the molecule throughan oxygen, nitrogen or sulfur atom, respectively. Similarly, the term“dialkylamino” is used in a conventional sense to refer to —NR′R″wherein the R groups can be the same or different alkyl groups.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon, or combinations thereof, fully saturated orcontaining from 1 to 3 degrees of unsaturation, consisting of the statednumber of carbon atoms and from one to three heteroatoms selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S may be placed atany interior position of the heteroalkyl group. The heteroatom Si may beplaced at any position of the heteroalkyl group, including the positionat which the alkyl group is attached to the remainder of the molecule.Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃—)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatomsmay be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃. Also included in the term “heteroalkyl” are thosegroups described in more detail below as “heterocycloalkyl.” The term“heteroalkylene” by itself or as part of another substituent means adivalent group derived from heteroalkyl, as exemplified by—CH₂—CH₂—S—CH₂CH₂— and —CH₂—S—CH₂CH₂—NH—CH₂—. For heteroalkylene groups,heteroatoms can also occupy either or both of the chain termini. Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied.

The term “acyl” refers to those groups derived from an organic acid byremoval of the hydroxy portion of the acid. Accordingly, acyl is meantto include, for example, acetyl, propionyl, butyryl, decanoyl, pivaloyl,benzoyl and the like.

An “activated carbonyl” group is a carbonyl group whose electrophilicityis enhanced as a result of the groups attached to either side of thecarbonyl. Examples of such activated carbonyl groups are(polyfluoroalkyl)ketones, (polyfluoroalkyl)aldehydes, alpha-keto esters,alpha-keto acids, alpha-keto amides, 1,2-diketones, 2-acylthiazoles,2-acylimidazoles, and the like.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “fluoroalkyl,” aremeant to include monofluoroalkyl and polyfluoroalkyl.

The term “aryl,” employed alone or in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) means, unless otherwise stated,an aromatic substituent which can be a single ring or multiple rings (upto three rings) which are fused together or linked covalently. The term“heteroaryl” is meant to include those aryl rings which contain fromzero to four heteroatoms selected from N, O, and S, wherein the nitrogenand sulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. The “heteroaryl” groups can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl ring systems are selected from the group of acceptable substituentsdescribed below. The term “arylalkyl” is meant to include those groupsin which an aryl or heteroaryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) or a heteroalkylgroup (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl,and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl” and “aryl”) aremeant to include both substituted and unsubstituted forms of theindicated group. Preferred substituents for each type of group areprovided below.

Substituents for the alkyl and heteroalkyl groups (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′, S(O)₂R′,—S(O)₂NR′R″, —CN and —NO₂ in a number ranging from zero to (2N+1), whereN is the total number of carbon atoms in such group. R′, R″ and R′″ eachindependently refer to hydrogen, unsubstituted(C₁-C₈)alkyl andheteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens,unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C₁-C₄)alkylgroups. When R′ and R″ are attached to the same nitrogen atom, they canbe combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similarly, substituents for the aryl groups are varied and are selectedfrom: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO₂, —CO₂R′,—CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)₂R′,—NR′—C(O)NR″R′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —NR″—S(O)₂—R′, —N₃, —CH(Ph)₂,perfluoro(C₁-C₄)alkoxy, and perfluoro(C₁-C₄)alkyl, in a number rangingfrom zero to the total number of open valences on the aromatic ringsystem; and where R′, R″ and R′″ are independently selected fromhydrogen, (C₁-C₈)alkyl and heteroalkyl, unsubstituted aryl,(unsubstituted aryl)-(C₁-C₄)alkyl, and (unsubstitutedaryl)oxy-(C₁-C₄)alkyl.

Two of the substituents on adjacent atoms of the aryl ring mayoptionally be replaced with a substituent of the formula-T—C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, —CH₂— or asingle bond, and the subscript q is an integer of from 0 to 2.Alternatively, two of the substituents on adjacent atoms of the arylring may optionally be replaced with a substituent of the formula-A-(CH₂)_(r)—B—, wherein A and B are independently —CH₂—, —NH—, —S(O)—,—S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integer of from 1 to3. One of the single bonds of the new ring so formed may optionally b ereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of the aryl ring may optionally be replaced with asubstituent of the formula —(CH₂)_(s)—X—(CH₂)_(t)—, where s and t areindependently integers of from 0 to 3, and X is —O—, —NR′—, —S(O)—,—S(O)₂—, or —S(O)₂NR′—. The substituent R′ in —NR′— and —S(O)₂NR′— isselected from hydrogen or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), and sulfur (S).

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds of Formula I, Formula II, Formula III, FormulaIV, Formula V, Formula VI, Formula VII, and/or Formula VIII that areprepared with relatively nontoxic acids or bases, depending on theparticular substituents found on the compounds described herein.Examples of pharmaceutically acceptable base addition salts include, butare not limited to, sodium, potassium, calcium, ammonium, organic amino,magnesium salt, or other similar salt. Examples of pharmaceuticallyacceptable acid addition salts include, but are not limited to, thosederived from inorganic acids like hydrochloric, hydrobromic, nitric,carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrophosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,oxalic, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like.

Small-Molecule Modulators of Trp-p8 Activity

Small-molecule Trp-p8 modulators that are suitably employed in thecompositions and methods of the present invention are exemplified hereinby the following Trop-p8 agonist compounds: Menthol (Sigma-Aldrich,Inc.; St. Louis, Mo.; (2-isopropyl-5-methyl-cyclohexanol)); Frescolat ML(Harris & Ford, LLC; Indianapolis, Ind.; Menthyl lactate); Frescolat MGA(Harris & Ford, LLC; Menthone Glycerin Acetal); L-Menthyl Acetate(Millennium Chemicals; Olympia Fields, Ill.;Cyclohexanol-5-methyl-2-(1-methylethyl)-acetate-[1R-(1alpha,2beta,5alpha)]-); L-Carvone (Millennium Chemicals;(R)-5-Isopropenyl-2-methyl-2-cyclohexenone p-Mentha-6,8-dien-2-one);WS-3 (Millennium Chemicals; N-Ethyl-p-menthane-3-carboxamide akacyclohexanecarboxamide, N-ethyl-5-methyl-2(1-methylethyl)); CoolingAgent 10 (Takasago International Corp.; Rockleigh, N.J.;(1)-Menthoxypropane-1,2-diol); Coolact P® (Takasago International Corp.;(−)-Isopulegol); Coolact 38D® (Takasago International Corp.); Icilin(1-(2-hydroxyphenyl)-4-(3-nitrophenyl)-1,2,3,6-tetrahydropyrimidine-2-oneaka3,4-dihydro-3-(2-hydroxyphenyl)-6-(3-nitrophenyl)-(1H)-pyrimidin-2-one);WS-23 (2-Isopropyl-N,2,3-trimethylbutyramide akaN,2,3-trimethyl-2-isopropyl butamide), and WS-12(N-(4-methoxyphenyl)-p-menthan-3-carboxamide aka cyclohexanecarboxamide,N-(4-methoxyphenyl)-5-methyl-2(1-methylethyl)).

The present invention further contemplates that additional Trp-p8agonists, including derivatives of the compounds of Formulas I, II, III,IV, V, VI, VII, and/or VIII disclosed herein, may also be suitablyemployed in the compositions and methods of the present invention.

Thus, in one embodiment, the present invention provides the followingTrp-p8 modulators and derivatives thereof:

A compound of Formula I

wherein

R¹ is selected from the group consisting of H, OH, CH₃, CH₃—CH—CH₃(isopropyl), and CH₃—C═CH₂ (isopropenyl);

R² is selected from the group consisting of H;

R³ is selected from the group consisting of O, OH, acetate, lactate,carboxamide, butanamide, sulphanamide, and propanetriol; and

R⁴ is selected from the group consisting of CH₃—CH—CH₃ (isopropyl),isopropane-2-ol, and CH₃—C═CH₂ (isopropenyl).

Exemplary compounds of Formula I include the following Trp-p8 agonists:

These exemplary Formula I Trp-p8 agonists have the following trade andchemical names: Menthol (2-isopropyl-5-methyl-cyclohexanol)(Sigma-Aldrich, Inc.; St. Louis, Mo.); Frescolat ML (Harris & Ford, LLC;Indianapolis, Ind.; Menthyl lactate); L-Menthyl Acetate (MillenniumChemicals; Olympia Fields, Ill.;Cyclohexanol-5-methyl-2-(1-methylethyl)-acetate-[1R-(1alpha,2beta,5alpha)]-); Cooling Agent 10 (Takasago International Corp.;Rockleigh, N.J.; (1)-Menthoxypropane-1,2-diol); Coolact P® (TakasagoInternational Corp.; (−)-Isopulegol); and Coolact 38D® (TakasagoInternational Corp.).

In another embodiment, the present invention further provides thefollowing small-molecule Trp-p8 modulators and derivatives thereof:

A compound of Formula II

wherein

R⁵ is selected from the group consisting of H, OH, CH₃, CH₃—CH—CH₃(isopropyl), and CH₃—C═CH₂ (isopropenyl);

R⁶ is selected from the group consisting of N;

R⁷ is selected from the group consisting of O and N;

R⁸ is selected from the group consisting of NH, O, and S; and

R⁹ is selected from the group consisting of NO₂.

Compounds of Formula II are exemplified herein by the Trp-p8 agonistIcilin(1-(2-hydroxyphenyl)-4-(3-nitrophenyl)-1,2,3,6-tetrahydropyrimidine-2-oneaka3,4-dihydro-3-(2-hydroxyphenyl)-6-(3-nitrophenyl)-(1H)-pyrimidin-2-one).

In another embodiment, the present invention further provides thefollowing acyclic carboxamide Trp-p8 modulators and derivatives thereofas presented in U.S. Pat. No. 4,153,679, incorporated herein byreference:

A compound of Formula III

wherein

R¹⁰ is selected from the group consisting of H and a C₁-C₅ alkylincluding, but not limited to, CH₃, C₂H₅, CH₃—CH—CH₃ (isopropyl), andCH₃—C═CH₂ (isopropenyl);

R¹¹ is selected from the group consisting of OH, carboxamide,butanamide, propanetriol, and CONR′R″, wherein R′ is selected from thegroup consisting of H, CH₃, C₂H₅, C₄H₈ (cyclobutyl), and C₄H₈O, andwherein R″ is selected from the group consisting of C₂H₅OOCH₂, C₂H₅,CH₃—CH—CH₃ (isopropyl), HOCH₂C(CH₃)₂, HOCH₂CH₂, C₄H₉ (tertbutyl), C₄H₉(secbutyl);

R¹² is selected from the group consisting of H and a C₁-C₅ alkylincluding, but not limited to, CH₃, CH₃—CH—CH₃ (isopropyl), andCH₃—C═CH₂ (isopropenyl), C₄H₉ (secbutyl), C₄H₉ (isobutyl), C₄H₉(n-butyl), C₅H₁₁ (isohexyl); and

R¹³ is selected from the group consisting of H and a C₁-C₅ alkylincluding, but not limited to, CH₃, C₂H₅, CH₃—CH—CH₃ (isopropyl),CH₃—C═CH₂ (isopropenyl), C₄H₉ (secbutyl), C₄H₉ (isobutyl).

Compounds of Formula III are exemplified herein by the Trp-p8 agonistWS-23 (2-Isopropyl-N,2,3-trimethylbutyramide akaN,2,3-trimethyl-2-isopropyl butamide).

In another embodiment, the present invention further provides thefollowing 3-substituted-p-menthane Trp-p8 modulators and derivativesthereof as presented in U.S. Pat. No. 4,150,052, incorporated herein byreference:

A compound of Formula IV

wherein

R¹⁴ is selected from the group consisting of H and an aliphatic groupcontaining up to 25 carbon atoms;

R¹⁵ is selected from the group consisting of H, OH and an aliphaticgroup containing up to 25 carbon atoms, with the proviso that when R¹⁵is H, R¹⁴ may also be an aryl group of up to 10 carbon atoms andselected from the group consisting of substituted phenyl, phenalkyl,substituted phenalkyl, naphthyl, substituted naphthyl, and pyridyl; and

R¹⁴ and R¹⁵, when taken together with the nitrogen atom to which theyare attached, may form a cyclic or heterocyclic group of up to 25 carbonatoms, e.g., a piperidino or a morpholino group. Exemplary such cyclicalgroups may be selected from the group consisting of3-phenyl-piperidin-1-yl, 3-phenyl-pyrrolidin-1-yl,6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-yl, and4-pyrimidin-2-yl-piperazin-1-yl.

Typical values for R¹⁴ and R¹⁵ when aliphatic are methyl, ethyl, propyl,butyl, isobutyl, n-decyl, cyclopropyl, cyclohexyl, cyclopentyl,cycloheptylmethyl, 2-hydroxyethyl, 3-hydroxy-n-propyl,6-hydroxy-n-hexyl, 2-aminoethyl, 2-acetoxyethyl, 2-ethylcarboxyethyl,4-hydroxybut-2-ynyl, and carboxymethyl.

When R¹⁴ is aryl, typical values are benzyl, naphthyl, 4-methoxyphenyl,2-methoxy-4-methoxyphenyl, 3-methoxy-5-methoxyphenyl,4-methyl-5-chlorophenyl, 4-hydroxyphenyl, 4-methylphenyl,3-methyl-4-methylphenyl, 3-hydroxy-4-methylphenyl, 4-fluorophenyl,4-chlorophenyl, 4-bromophenyl, 4-ethylphenyl, 2-fluoro-4-fluorophenyl,4-nitrophenyl, 2-hydroxynaphthyl, pyridyl,[1-carbamoyl-2-(1H-indol-3-yl)-ethyl,1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl,1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl,1-carbamoyl-2-(4-hydroxyphenyl)ethyl, 1-carbamoyl-2-phenylethyl,1-hydroxymethyl-2-(1H-indol-3-yl)ethyl,1-hydroxymethyl-2-(4-chlorophenyl)ethyl,1-hydroxymethyl-2-(4-hydroxyphenyl)ethyl,1-hydroxymethyl-2-hydroxy-2-phenylethyl, 1-hydroxymethyl-2-phenyl ethyl,1-methoxymethyl-2-phenylethyl, 1-methyl-2-(4-chlorophenyl)-ethyl,1-methyl-2-(5-fluoro-1H-indol-3-yl)-ethyl,1-methyl-2-hydroxy-2-phenylethyl, 1-oxo-2-phenylethyl,1-phenyl-cyclopentylmethyl, 2-(1-cyclopentyl-pyrrolidin-3-yl)-ethyl,2-(1H-indol-3-yl)ethyl, 2-(2,3-dimethoxyphenyl)ethyl,2-(2,4-dichlorophenyl)ethyl, 2-(2,4-dimethylphenyl)ethyl,dimethoxyphenyl)ethyl, 2-(2,5-dimethylphenyl)-ethyl,2-(2,6-dimethylphenyl)ethyl, 2-(2-chloro-6-fluorophenyl)ethyl,2-(2-chlorophenyl)ethyl, 2-(2-fluorophenyl)ethyl, 2-(2-furyl)ethyl,2-(2-methoxy-5-bromophenyl)ethyl, 2-(2-methoxyphenyl)-ethyl,2-(2-methylphenyl)ethyl, 2-(3,4-dichlorophenyl)ethyl,2-(3,4-dimethoxyphenyl)ethyl, dimethoxyphenyl)ethyl,2-(3-bromo-4-methoxyphenyl)ethyl, 2-(3-chlorophenyl)ethyl,2-(3-ethoxyphenyl)ethyl, 2-(3-fluorophenyl)ethyl,2-(3-hydroxy-4-methoxyphenyl)-ethyl, 2-(3-hydroxyphenyl)ethyl,2-(3-methoxy-4-ethoxyphenyl)ethyl, 2-(3-methoxy-4-hydroxyphenyl)ethyl,2-(3-methoxyphenyl)ethyl, 2-(3-methylphenyl)ethyl,2-(3-trifluoromethylphenyl)ethyl, 2-(4-bromophenyl)-ethyl,2-(4-chlorophenyl)ethyl, 2-(4-ethylphenyl)ethyl,2-(4-fluorophenyl)ethyl, 2-(4-hydroxyphenyl)ethyl,2-(4-methoxy-phenyl)-2-oxo-ethyl, 2-(4-methoxyphenyl)ethyl,2-(4-methylphenyl)ethyl, 2-(4-methylphenyl)ethyl,2-(4-methylsulfanylphenyl)ethyl, 2-(4-nitrophenyl)ethyl,2-(4-sulfamoyl-phenyl)-ethyl, 2-(5-methoxy-1H-indol-3-yl)-ethyl,2-(6-fluoro-1H-indol-3-yl)-ethyl, 2-(6-methoxy-1H-indol-3-yl)-ethyl,2-(7-methyl-1H-indol-3-yl)-ethyl, 2-(N,N-dipropylamino)ethyl,2-(pyridin-2-yl)-ethyl, 2-(pyridin-3-yl)-ethyl, 2-(pyridin-4-yl)-ethyl,2,2-diphenylethyl, 2,3-difluorobenzyl, 2,3-dimethoxyphenyl,2,4-dichlorophenyl, 2,4-difluorophenyl, 2,4-dimethoxyphenyl,2,4-dimethylphenyl, 2-bromo-4-methylphenyl, 2-chloro-4-cyanophenyl,2-chloro-4-fluorophenyl, 2-chloro-4-iodophenyl, 2-chloro-4-nitrophenyl,2-chloro-5-nitrophenyl, 2-chlorophenyl, 2-cyclohex-1-enyl-ethyl,2-fluoro-4-chlorophenyl, 2-fluoro-5-nitrophenyl,2-hydroxy-2-(3-hydroxyphenyl)ethyl, 2-hydroxy-2-(4-hydroxyphenyl)ethyl,2-hydroxy-2-phenylethyl, 2-iodophenyl,2-methyl-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl, 2-methyl-2-phenylethyl,2-methyl-4-bromophenyl, 2-methyl-5-nitrophenyl, 2-methylphenyl,2-nitro-4-fluorophenyl, 2-nitrophenyl,2-oxo-2,3-dihydro-1H-benzoimidazol-5-yl, 2-oxo-2-phenylethyl,2-phenyl-1H-benzoimidazol-5-yl, 2-phenyl-2-(4-chlorophenyl)ethyl,2-phenyl-2-(4-fluorophenyl)-ethyl, 2-phenyl-2-(4-methoxyphenyl)ethyl,2-phenylethenyl, 2-phenylethyl, 2-pyridin-2-yl-benzooxazol-5-yl,2-pyridin-3-yl-1H-benzoimidazol-5-yl, 2-thiophen-2-yl-ethyl),2-trifluoromethyl-1H-benzoimidazol-5-yl, 3,4,5-trifluorophenyl,3,4,5-trimethoxyphenyl, 3,4-cyclopentanephenyl, 3,4-dichlorophenyl,3,4-dimethylphenyl, 3,5-dimethoxyphenyl, 3-acetamidophenyl,3-bromo-4-methylphenyl, 3-carboxamidophenyl, 3-chloro-4-methoxyphenyl,3-chloro-4-methylphenyl, 3-chloro-4-morpholin-4-yl-phenyl,3-hydroxymethylphenyl, 3-nitrophenyl, 3-oxo-indan-5-yl, 3-phenylpropyl,3-yl-acetophenone, 4-(1,1-dioxo-1l6-thiomorpholin-4-ylmethyl)-phenyl,4-(1-hydroxyethyl)phenyl, 4-(2-hydroxyethyl)phenyl,4-(4-ethyl-piperazin-1-yl)-phenyl,4-(4-methyl-1H-benzoimidazol-2-yl)-phenyl,4-(4-methyl-piperazin-1-ylmethyl)-phenyl,4-(morpholine-4-sulfonyl)-phenyl,4-[1,3-dioxo-2-(2-trifluoromethyl-phenyl)-2,3-dihydro-1H-isoindol-5-yloxy]-phenyl,4-[2-(2-methoxy-phenyl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yloxy]-phenyl,4-{2-[2-(3,4-dimethoxy-phenyl)-ethyl]-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yloxy}-phenyl,4-acetylphenyl, 4-azepan-1-ylmethyl-phenyl, 4-benzooxazol-2-yl-phenyl,4-Biphenyl, 4-bromophenyl, 4-carboxamidophenyl, 4-chlorophenyl,4-cyanophenyl, 4-ethoxyphenyl, 4-ethylphenyl, 4-fluorophenyl,4-hydroxymethylphenyl, 4-methoxyphenyl, 4-methyl-2-oxo-2H-chromen-7-yl,4-methylcyclohexyl, 4-methylphenyl, 4-methylsulfanylphenyl,4-nitrobenzyl, 4-pyrrolidin-1-ylmethyl-phenyl, 4-trifluoromethylphenyl,benzo[1,3]dioxol-5-yl, benzoylamino, benzyloxy, bicyclo[2.2.1]hept-2-yl,C-1H-indazol-5-yl, cycloheptyl, indan-2-yl,N-(2-diethylamino-ethyl)-benzamide-4-yl, N′-quinoxalin-2-yl-amino, andphenylcyclopropyl.

Trp-p8 modulators of Formula IV are exemplified herein by the Trp-p8agonists WS-3 (Millennium Chemicals; N-Ethyl-p-menthane-3-carboxamideaka cyclohexanecarboxamide, N-ethyl-5-methyl-2(1-methylethyl)) and byWS-12 (N-(4-methoxyphenyl)-p-menthan-3-carboxamide akacyclohexanecarboxamide, N-(4-methoxyphenyl)-5-methyl-2(1-methylethyl)),and the compounds presented in Table 1.

In a further embodiment, the present invention provides the followingTrp-p8 modulators and derivatives thereof that comprise at least oneketal moiety, including, but not limited to the Trp-p8 agonists1-menthone glycerol ketal and 3,3,5-trimethylcyclohexaone glycerol ketalpresented in U.S. Pat. No. 5,266,592, incorporated herein by reference:

A compound comprising at least one ketal of Formula V

wherein

R¹⁶ is selected from the group consisting of a C₂-C₆ alkylene grouphaving at least one, but not more than 3, hydroxyl group(s), preferably1 hydroxyl group, and

Either R¹⁷ and R¹⁸ independently of one another represent C₁-C₁₀-alkylwhich is optionally substituted by 1 to 3 groups selected from the groupconsisting of hydroxyl, amino, thio, and halogen (e.g., fluorine,chlorine, bromine, or iodine), C₅-C₇-cycloalkyl, preferably cyclohexyl,C₆-C₁₂-aryl, preferably phenyl, with the proviso that the total of the Catoms of R¹⁷ and R¹⁸ is not less than 3, or

R¹⁷ and R¹⁸ together represent an alkylene group that, together with thecarbon atom that carries the groups R¹⁷ and R¹⁸, forms a 5-7-memberedring, it being possible for this alkylene group, in turn, to besubstituted by C₁-C₆-alkyl groups.

Preferred groups R¹⁷ and R¹⁸ comprise methyl, isopropyl, and tert-butyl.

Compounds comprising a ketal of Formula V include the following Trp-p8modulator compounds wherein R¹⁶ is as defined above:

Compounds comprising a ketal of Formula V are exemplified herein by theTrp-p8 agonist Frescolat MGA (Harris & Ford, LLC; Menthone GlycerinAcetal),

In yet a further embodiment, the present invention provides Formula VIsmall-molecule Trp-p8 modulators and derivatives thereof exemplifiedherein by L-Carvone (Millennium Chemicals;(R)-5-Isopropenyl-2-methyl-2-cyclohexenone p-Mentha-6,8-dien-2-one),

Still further embodiments of the present invention providesmall-molecule antagonists of the small-molecule Trp-p8 agonistspresented herein. Thus, within certain embodiments are provided FormulaI, Formula II, Formula III, Formula IV, Formula V, Formula VI, FormulaVII, and/or Formula VIII small-molecule antagonists, and derivativesthereof, of the Formula I, Formula II, Formula III, Formula IV, FormulaV, Formula VI, Formula VII, and/or Formula VIII Trp-p8 agonistsdisclosed herein above.

Presented herein in the Examples are specific Trp-p8 antagonists thatare exemplary of the Trp-p8 modulators of Formula VII

wherein

R¹⁷ is selected from the group consisting of 2-pyridyl,2-nitro-4-trifluoromethylphenyl, 2-nitro-4-chlorophenyl,2-methoxyphenyl, 2-chlorophenyl, phenyl, 2-methyl-quinolin-3-yl,4-methoxyphenyl, 4-fluorophenyl,3-azepan1-yl-5-(4-trifluoromethoxy)phenylamino[1,3,5]triazyl,cyclohexyl, diphenylmethyl, 2-phenylethyl, 4-hydroxy-cyclohexyl,cycloheptyl, cyclopentyl, C-benzo[1,3]dioxol-5-yl-methyl, 2-pyridyl, and4-chlorobenzyl;

R¹⁸ is selected from the group consisting of1-benzyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl, 3-benzylamino-2-nitrophenyl,5-nitro-quinolin-8-yl,1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol,1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,benzyl-2-methyl-quinazolin-4-yl,3-methyl-5-morpholin-4-yl-2-nitro-phenyl,2-nitro-5-piperazin-1-yl-ethanol,1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol,4-(2,5-dimethyl-pyrrol-1-yl)-2-nitro-phenyl,2-nitro-3-trifluoromethanesulfonyl-phenyl,1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,2-(2-Fluoro-phenoxymethyl)-2-cyano oxazolyl, adamantly,5-(benzo[1,3]dioxol-5-ylamino)-10b,10c-dihydro-anthra[1,9-cd]isoxazol-6-one-yl,2-methyl-thiazolo[3,2-b][1,2,4]triazol-6-ol 4-methylphenyl methyl,3-benzyl-3H-quinazolin-4-one-2-yl, cyclopentyl, tetrahydronapthyl,cyclooctyl, cyclohexyl,C-[3-(4-chloro-phenyl)-2,5-dimethyl-pyrazolo[1,5-a]pyrimidin-7-yl]-methyl,C-(2-benzyl-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-methyl,and 1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol;

R¹⁹ and R²⁰ are each independently selected from the group consisting ofH and O; and

R²¹ is selected from the group consisting of 4-methylphenyl,2-chloro-4-fluorophenyl, and 4-chlorophenyl.

In other embodiments, the present invention provides Formula VIIIsmall-molecule modulators, and derivatives thereof, including FormulaVIII antagonists of the Formula IV Trp-p8 agonists disclosed hereinabove and in the Examples.

A compound of Formula VIII

wherein

R²² is a linker moiety, which may be selected from the group consistingof oxyacetamide, urea, carbamate, thiourea, sulfonamide, amine, amide.Formula VIII antagonists are represented by the following sub-formulae(Formula VIII-A, Formula VIII-B, Formula VIII-C, Formula VIII-D, FormulaVIII-E, Formula VIII-F, and Formula VIII-G):

Irrespective of which of the seven R²² linker moieties is employed, R²³is selected from the group consisting of H, tetrahydro isoquinolinyl,tetrahydro quinolinyl, 3-methyl indolinyl, indolinyl, 2-(N-methyl,N-phenylethyl)amino ethyl, 3-methyl indolinyl, 1-phenyl ethyl, 2-chlorobenzyl, 2-methoxybenzyl, 2-methoxyphenyl, 2-cyclohex-1-enyl ethyl,(1-phenyl-cyclophenyl)-methyl, 2-(tetrahydroquinolinyl)-ethyl,1-propyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, cycloheptyl,3-cyclohexylsulfanylpropyl, 2-cyclohex-1-enyl ethyl,2-(N-isopropyl,N-phenylethyl)amino ethyl,1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, 2-cyclopentylethyl,2-phenylcyclopropyl, 1-phenoxyethyl, 4-butyloxyphenyl,(2-nitrophenoxy)methyl,4,7,7-trimethyl-2-oxa-bicyclo[2.2.1]heptan-3-one,C-(1-phenyl-5-propyl-1H-pyrazol-4-yl)-methyl, benzyl, 2-chlorobenzyl,1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-methoxy-phenyl]-2,3,4,9-tetrahydro-1H-b-carboline,C-[3-(4-butoxy-phenyl)-1H-pyrazol-4-yl]-methyl,4-(azepane-1-sulfonyl)-phenyl, and547-chloro-quinolin-4-ylsulfanyl)-[1,3,4]thiadiazol-2-yl;

R²⁴ is selected from the group consisting of H, tetrahydroisoquinolinyl, tetrahydro quinolinyl, 3-methyl indolinyl, indolinyl,3-methyl indolinyl, 1-propyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine,1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, and1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-methoxy-phenyl]-2,3,4,9-tetrahydro-1H-b-carboline;and

R²⁵ is selected from the group consisting of H.

Synthesis of Small-Molecule Trp-p8 Modulators

As noted above, compounds of the present invention include compounds ofFormula I, Formula II, Formula III, Formula IV, Formula V, Formula VI,Formula VII, and Formula VIII. Within certain aspects, compounds of thepresent invention can be obtained from commercial sources and/or can bemade using commercially available starting materials by employingsynthetic methodology readily available in the art. Compounds ofFormulae I-VIII may be isolated using typical isolation and purificationtechniques known in the art, including, for example, chromatographic andrecrystallization methods.

Exemplary compositions and methodology for their synthesis are disclosedin the following patents, each of which is incorporated herein byreference: U.S. Pat. No. 4,150,052 discloses N-substituted p-menthane3-carboxyamide compounds having a physiological cooling action on theskin; U.S. Pat. No. 4,153,679 discloses compositions comprising acyclictertiary and secondary carboxamides that have a physiological coolingaction on the skin; U.S. Pat. No. 4,020,153 discloses cyclicsulphonamides and sulphinamides having a physiological cooling action onthe skin; European Patent Application No. 1 157 617 discloses alpha-ketoenamine derivatives in a variety of food, cosmetic, pharmaceutical, andperfume compositions; U.S. Pat. No. 4,296,093 discloses alkylsubstituted cyclohexanamides having a physiological cooling effect onthe skin; U.S. Pat. No. 5,756,857 discloses cyclohexanol derivativeshaving a cool feeling; U.S. Pat. No. 4,248,859 discloses alicyclicamides having a physiological cooling effect; U.S. Pat. No. 5,266,592discloses ketals, such as glycerol ketals, for example 1-menthoneglycerol ketal or 3,3,5-trimethylcyclohexanone glycerol ketal, that havea physiological cooling effect; U.S. Pat. No. 6,328,982 disclosescompositions comprising a cooling compound such as 1-menthol(2-isopropyl-5-methyl-cyclohexanol), 1-isopulegol,3-(1-methoxy)propane-1,2-diol and p-menthane-3,8-diol; and U.S. Pat. No.4,459,425 discloses 3-1-menthoxypropane-1,2-diol and its associatedcooling activity.

Those of skill in the art will readily recognize that compounds suitablyincluded in the compositions and methods of the present invention canexist in a number of cis and trans isomers, E/Z forms, diastereomers, aswell as optical isomers. Thus, compounds used in the compositions andmethods of the present invention include all such combinations andvariations.

In compounds of Formula I, Formula II, Formula III, Formula IV, FormulaV, and Formula VI carbon atoms to which four non-identical substituentsare bonded are asymmetric. Accordingly, compounds of Formula I, FormulaII, Formula III, Formula IV, Formula V, or Formula VI may exist asenantiomers, diastereomers or a mixture thereof. The enantiomers anddiastereomers may be separated by chromatographic or crystallizationmethods, or by other methods known in the art. The asymmetric carbonatom may be in one of two configurations, R or S, both of which arewithin the scope of the present invention. The presence of small amountsof the opposing enantiomer or diastereomer in the final purified productdoes not affect the therapeutic application of such compounds.

Compounds of Formulae I-VI may be further treated to formpharmaceutically acceptable salts. Treatment of a compound of theinvention with an acid or base may form, respectively, apharmaceutically acceptable acid addition salt and a pharmaceuticallyacceptable base addition salt, each as defined above. Various inorganicand organic acids and bases known in the art, including those describedherein above, may be used to effect the conversion to the salt.

The present invention also relates to pharmaceutically acceptableisomers, hydrates, and solvates of compounds of Formula I, Formula II,Formula III, Formula N, Formula V, and Formula VI. Compounds of theseformulae may also exist in various isomeric and tautomeric formsincluding pharmaceutically acceptable salts, hydrates and solvates ofsuch isomers and tautomers.

This invention also encompasses prodrug derivatives of the compounds ofFormula I, Formula II, Formula III, Formula N, Formula V, and FormulaVI. The term “prodrug” refers to a pharmacologically inactive derivativeof a parent drug molecule that requires biotransformation, eitherspontaneous or enzymatic, within the organism to release the activedrug. Prodrugs are variations or derivatives of the compounds ofFormulae I-VI of the present invention that have groups cleavable undermetabolic conditions. Prodrugs become the compounds of the inventionwhich are pharmaceutically active in vivo when they undergo solvolysisunder physiological conditions or undergo enzymatic degradation. Anexemplary prodrug technology that may be suitably employed with thecompounds of the present invention is the protease activated cancertherapy (PACT) technology described in detail within U.S. patentapplication Ser. No. 10/156,214 and PCT Application Publication No. WO02/095007, both of which are incorporated herein by reference.

Synthesis of compounds of Formula I, may be achieved, as described belowin reference to compounds of Formula IV, by reacting an acid chloride,obtained by reacting p-menthane-3-carboxylic acid with thionyl chloride,with the appropriate amine. As noted below, typically, the reaction iscarried out in solution at room temperature in the presence of ahydrogen chloride receptor (e.g., sodium hydroxide).

Synthesis of (1)-Menthoxypropane-1,2-diol (Cooling Agent 10) from1-menthol (2-isopropyl-5-methyl-cyclohexanol) is described in U.S. Pat.No. 4,459,425, incorporated herein by reference. Briefly, 1-menthol andmetallic sodium or sodium hydride are introduced into a solvent (e.g.,toluene or xylene) and heated. When the temperature reaches 100° C., orhigher, the reaction starts and generation of hydrogen gas occurs. Afterconfirming that the generation of hydrogen has stopped, the mixture isfurther heated at the reflux temperature of the solvent to complete thereaction.

Allyl halide (e.g., allyl chloride or allyl bromide) is then added tothe reaction mixture in small portions. As the reaction proceeds, sodiumhalide deposits and the reaction solution becomes slurry-like. After thereaction is completed, the reaction solution is cooled, and afteraddition of water, the resulting mixture is stirred. Then, a solvent,(e.g., benzene, toluene, ether, hexane, or petroleum ether) is added.The organic layer is separated and washed with saturated saline water.After the solvent is recovered, the residue is distilled under reducedpressure to obtain 3-1-menthoxypropate-1-ene.

The 3-1-menthoxypropane-1-ne is oxidized into the corresponding oxide byuse of an organic peracid. The oxide is hydrolyzed to form the desired3-1-methoxypropane-1,2-diol. That is, an organic acid, (e.g., formicacid or acetic acid) and aqueous hydrogen peroxide are mixed with3-1-menthoxypropane-1-ene and gradually heated carefully while stirring.The organic acid and the hydrogen peroxide react, forming an organicperacid that participates in the oxidation reaction. The reaction isexothermic, and rapid heating should be avoided. When the temperature ofthe reaction solution reaches near 50° C., the heating is stopped. Itis, thereafter, necessary for the temperature of the reaction solutionto be maintained at about 70° C. by external cooling to prevent afurther temperature increase caused by the heat of reaction. If thetemperature of the reaction solution is excessively high, the organicperacid decomposes before it participates in the oxidation reaction,resulting in a reduction in yield.

After the reaction is completed, a solvent (e.g., benzene, toluene,xylene, or petroleum ether) is added to perform the extraction. Theextracted liquid is washed with water. Upon recovery of the solvent bydistillation, a crude oxide in the form of an organic acid ester isproduced. The crude oxide thus formed is mixed with an about 20% aqueoussolution or caustic soda, for example, and is hydrolyzed by boiling forabout 1 hour to produce the desired 3-1-menthoxypropane-1,2-diol.

Synthesis of compounds of Formula II, exemplified herein by1-(2-hydroxyphenyl)-4-(3-nitrophenyl)-1,2,3,6-tetrahydropyrimidine-2-one(Icilin aka AG-3-5), is disclosed in U.S. Pat. No. 3,821,221,incorporated herein by reference. Briefly,β-diethlyamino-m-nitropropiophenone hydrochloride is added to 50%aqueous ethanol and the mixture refluxed with stirring until thehydrochloride is dissolved. O-aminophenol is added and the solutionrefluxed for 30 minutes, then set aside to cool. The reaction product iscrystallized out of solution to yield(3-(o-hydroxyanilino)-m-nitrophropiophenone (mp of 107° C. to 109° C.).β-(o-hydroxyanilino)-m-nitrophropiophenone is dissolved in ethanol andconcentrated HCl is added to acidify the solution. The solvent isevaporated in vacuo and β-(o-hydroxyanilino-m-nitropropiophenone)-HCl(mp of 172° C. to 173° C.) is crystallized from methanol-acetone.β-(o-hydroxyanilino-m-nitropropiophenone)-HCl is dissolved in aceticacid at 60° C. Potassium cyanate is added and the reaction mixturecooled to room temperature. Water is added and the crystalline1-(2-hydroxyphenyl)-4-(3-nitrophenyl)-1,2,3,6-tetrahydropyrimidine-2-one(mp of 228° C. to 230° C.) is recovered by filtration.

Synthesis of acyclic carboxamide compounds of Formula III, asexemplified herein by 2-isopropyl-N,2,3-trimethylbutyramide akaN,2,3-trimethyl-2-isopropyl butamide (WS-23), is disclosed in U.S. Pat.No. 4,153,679, incorporated herein by reference. Briefly, Formula IIIamides may be prepared by conventional techniques known to those ofskill in the art, for example, by reacting an acid chloride of theformula R₁₀R₁₂R₁₃COCl with an amine (R₁₁), as indicated above, in thepresence of hydrogen chloride acceptor.

N,2,3-trimethyl-2-isopropyl butamide may be prepared, for example, byheating 2,3-Dimethyl-2-isopropylbutanic acid under reflux with thionylchloride for 60 minutes. The excess of thionyl chloride may be removedunder reduced pressure and the 2,3-dimethyl-2-isopropylbutanoyl chloridedistilled, bp. 73° C.-75° C./15 mm.

The acid chloride in ether may be added dropwise to a stirred solutionof methylamine (70% soln. in water) in ether with stirring. The etherlayer may then be washed with water, dilute HCl and water. The dried(MgSO₄) ether solution was concentrated, and the residue distilled togive N,2,3-trimethyl-2-isopropyl butamide (mp 58° C.-61° C., bp. 83°C.-85° C./0.35 mm.).

Synthesis of exemplary 3-substituted-p-menthane compounds that may besuitably employed as Trp-p8 modulators in the compositions and methodsof the present invention is described in U.S. Pat. No. 4,150,052,incorporated herein by reference in its entirety. For example, thecorresponding acid chloride (obtained by reactingp-menthane-3-carboxylic acid with thionyl chloride) may be reacted withthe appropriate amine. The reaction will usually be carried out at roomtemperature in solution in the presence of a hydrogen chloride receptor,e.g., sodium hydroxide.

The basic p-menthane structure is a chair-shaped molecule that can existin cis or trans form. Substitution of the carboxyl or amide group intothe 3-position gives rise to four configurational or geometric isomersdepending upon whether the substitution is axially or equatorially intothe cis or trans isomer, the four isomers are related as menthol is toneomenthol, isomenthol, and neoisomenthol.

In an exemplary reaction protocol, p-Menthane-3-carboxylic acid isheated under reflux with thionyl chloride. Excess thionyl chloride isdistilled off in vacuo. The crude p-menth-3-oyl chloride is dissolved indiethyl ether and the ethereal solution added with stirring and coolingto a solution of ethylamine and sodium hydroxide in water. The mixtureis stirred and the ethereal layer separated. The aqueous layer is washedwith ether and the combined ethereal solution washed with dilutehydrochloric acid and water. The ether solution is dried with MgSO₄ andevaporated to give a white crystalline solid. The solid isrecrystallised from acetone:water (9:1) by dissolving the crystals atroom temperature and then cooling to produceN-ethyl-p-menthane-3-carbozamide as a white crystalline solid, mp. 82.5°C.-84.5° C. Substitution of the amide group in the 3-position of thep-menthane structure gives rise to optical and geometric isomerism.

When either R¹³ or R¹⁴ is aliphatic, the preferred values are C₁-C₉straight or branched chain alkyl, C₁-C₉ straight or branched chainhydroxyalkyl or aminoalkyl and C₁-C₄ acylated derivatives thereof, and—C_(n)H_(2n)COR¹⁵ or —C_(n)H_(2n)COOR¹⁵, where —C_(n)H_(2n) is astraight or branched chain alkylene in which n is an integer of from 1-6and R¹⁵ is H or a C₁-C₈ alkyl or hydroxyalkyl group, preferably a C₁-C₄straight chain alkyl group.

When R¹³ is H and R¹⁴ is OH or substituted phenyl, e.g., alkylphenyl,hydroxyphenyl, alkoxyphenyl, halophenyl of up to 10 carbon atoms,phenalkyl or substituted phenalkyl, e.g., benzyl, naphthyl, orsubstituted naphthyl, and compounds where R¹³ and R¹⁴ are joined to forma cyclic group. When so joined, R¹³ and R¹⁴ preferably represent analkylene chain, optionally interrupted by oxygen, which together withthe nitrogen atom to which R¹³ and R¹⁴ are attached forms a 5- or6-membered heterocyclic ring.

Synthesis of exemplary compounds comprising one or more ketal, includingfor example menthone glycerine ketals, that may be suitably employed asTrp-p8 modulators in the compositions and methods of the presentinvention is described in U.S. Pat. No. 5,266,592, incorporated hereinby reference in its entirety.

For example, ketals of Formula V may be prepared by an acid-catalysedreaction of a ketone on which the ketal of Formula V is based and notless than the equivalent amount of aliphatic C3-C6-alcohol having notless than 3 and not more than 5, preferably 3, hydroxyl groups. Theketone on which the ketal of Formula V is based and an excess amount ofthe C3-C6 alcohol having 3 to 5 hydroxyl groups will be employed.

Exemplary acid catalysts that can be used are p-toluenesulphonic acid,phosphoric acid, or potassium hydrogen sulphate in catalyticallyeffective amounts. The reaction will generally be carried out either inan organic solvent that together with water forms an azeotrope, so thatthe water, which is liberated during formation of the ketal, can beeliminated by azeotropic entrainment or water-consuming co-reagents suchas, for example, trialkyl ortho esters are used. Exemplary organicsolvents include benzene, toluene, xylene, chloroform, methylenechloride and trichloroethylene. The reaction is complete when water nolonger separates out or when the ester/alcohol mixture is no longerseparated out. The products may be washed subsequently with dilutealkali and with water, to separate and dry the organic phase, to stripoff the solvent and, if appropriate, to purify the residue, for exampleby distillation.

An exemplary compound comprising a ketal of Formula V is 1-menthoneglycerol ketal (Frescolat MGA, aka menthone glycerin acetal; Harris &Ford, LLC), which is synthesized as follows. 2 mol of 1-menthone, 3 molof glycerol, and 5 g of potassium hydrogen sulphate are mixed in a 2liter three-neck flask in the presence of toluene. This mixture isrefluxed in a water separator. After 7 hours, water separates and themixture is neutralized and distilled.

Synthesis of the exemplary Formula VI terpene compound L-Carvone(2-cyclohexen-1-one, 2-methyl-5-(1-methylethenyl)-(R); MillenniumChemicals) from D-limonene is described in Ikan, Natural Products—ALaboratory Guide pp. 151-155 (Academic Press, 1969), incorporated hereinby reference. Briefly, a solution of D-limonene in isopropanol is cooledto below 10° C. Solutions of HCl in isopropanol and concentrated aqueoussodium nitrite are added dropwise to the D-limonene solution to generatelimonene nitrosochloride. The limonene nitrosochloride anddimethylformamide is refluxed with isopropanol. After cooling,crystallization is induced and the precipitate filtered and washed withwater. The resulting L-carvoxime is refluxed with 0.5 M oxalic acid andthe mixture is steam distilled. The distillate is extracted with ether,dried over anhydrous magnesium sulfate, and the excess ether evaporatedto leave L-carvone.

Additional synthetic methods for the preparation of Trp-p8 modulators ofthe present invention are presented herein in Examples 1-5.

Compositions Comprising a Small-Molecule Trp-p8 Modulators

As discussed above, the present invention is directed to small-moleculeTrp-p8 modulators, including Trp-p8 agonists and Trp-p8 antagonists thatbind to and alter the activity of Trp-p8. Within certain embodiments,Trp-p8 modulators are agonists that are, in certain instances, capableof stimulating cation influx in, and toxicity of, a cell expressing theTrp-p8 channel protein. Within alternative embodiments, Trp-p8modulators are antagonists of Trp-p8 activity that are capable ofreducing the activity of Trp-p8 expressed in a cell. Thus, Trp-p8modulators of the present invention will find utility in compositions,including pharmaceutical compositions, which are useful in the treatmentof diseases associated with Trp-p8 expression. Suitable compositions,according to the present invention, comprise one or more Trp-p8 agonistof Formula I, Formula II, Formula III, Formula IV, Formula V, FormulaVI, Formula VII, and/or Formula VIII and/or one or more Trp-p8antagonist of Formula I, Formula II, Formula III, Formula IV, Formula V,Formula VI, Formula VII, and/or Formula VIII, as described above, incombination with one or more pharmaceutically acceptable carrier orexcipient.

In one embodiment, the present invention provides small-molecule Trp-p8modulators in combination with a pharmaceutically acceptable excipientsuch as sterile saline or other medium, water, gelatin, oil, etc., toform pharmaceutically acceptable compositions. The compositions and/oragonists may be administered alone or in combination with any convenientcarrier, diluent, etc. and such administration may be provided in singleor multiple dosages. Useful carriers include, but are not limited to,solid, semi-solid, or liquid medium including water and non-toxicorganic solvents.

Pharmaceutical compositions of the present invention may be prepared bymixing one or more Trp-p8 agonist of Formula I, Formula II, Formula III,Formula IV, Formula V, Formula VI, Formula VII, and/or Formula VIII witha pharmaceutically acceptable carrier or agent. Alternatively,pharmaceutical compositions may be prepared by mixing one or more Trp-p8antagonist of Formula I, Formula II, Formula III, Formula IV, Formula V,Formula VI, Formula VII, and/or Formula VIII with a pharmaceuticallyacceptable carrier or agent. In addition, pharmaceutical compositionsmay further include excipients, stabilizers, diluents and the like andmay be provided in sustained release or timed release formulations.Acceptable carriers, agents, excipients, stabilizers, diluents and thelike for therapeutic use are well known in the pharmaceutical field, andare described, for example, in “Remington's Pharmaceutical Sciences,”(Mack Publishing Co., ed. A.R. Gennaro, 1985), incorporated herein byreference. Such materials are nontoxic to the recipients at the dosagesand concentrations employed and include buffers such a s phosphate,citrate, acetate, and other organic acid salts, antioxidants such asascorbic acid, low molecular weight peptides such as polyarginine,proteins, such as serum albumin, gelatin, or immunoglobulin, hydrophilicpolymers such as serum albumin, gelatin, or immunoglobulin, hydrophilicpolymers such as polyvinylpyrrolidinone, amino acids such as glycine,glutamic acid, aspartic acid, or arginine, monosaccharides,disaccharides, and other carbohydrates including cellulose or itsderivatives, glucose, mannose or dextrins, chelating agents such asEDTA, sugar alcohols such as mannitol or sorbitol, counterions such assodium and/or nonionic surfactants such as TWEEN, or polyethyleneglycol.

Within still further aspects, the compositions of the present inventioncomprise a compound of Formula I, Formula II, Formula III, Formula IV,Formula V, Formula VI, Formula VII, and/or Formula VIII formulatedtogether with one or more cancer therapeutic agent. Alternatively, thecompositions of the present invention comprise a compound of Formula I,Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII,and/or Formula VIII independently formulated with one or more cancertherapeutic agent. That is, the compound of Formula I, Formula II,Formula III, Formula IV, Formula V, Formula VI, Formula VII, and/orFormula VIII and the cancer therapeutic agent are separately formulated.

Suitable cancer therapeutic agents include, but are not limited to,antimitotic agents including, but not limited to, paclitaxel,vincristine, and etoposide; alkylating agents including, but not limitedto, mechlorethamine, cyclophosphamide, and carmustine; antimetabolitesincluding, but not limited to, methotrexate, gemcitabine, lometrexol,5-fluorouracil, and 6-mercaptopurine; cytotoxic antibiotics including,but not limited to, doxorubicin, daunorubicin, bleomycin, mitomycin C,and streptozocin; platinum agents including, but not limited to,cisplatin and carboplatin; hormonal agents including, but not limitedto, anti-estrogens such as tamoxifen and diethylstilbestrol as well asanti-androgens such as flutamide; antiangiogenesis agents; and farnesyltransferase inhibitors.

In certain aspects, compounds of Formula I, Formula II, Formula III,Formula IV, Formula V, Formula VI, Formula VII, and/or Formula VIII areadministered in combination with a cancer therapeutic agent that isineffective in stimulating Trp-p8-mediated cation influx.

In other aspects, compounds of Formula I, Formula II, Formula III,Formula IV, Formula V, Formula VI, Formula VII, and/or Formula VIE areadministered in combination with one or more additional Trp-p8 modulatorincluding, but not limited to a compound of Formula I, Formula II,Formula III, Formula IV, Formula V, Formula VI, Formula VII, and/orFormula VIII.

Depending upon the particular treatment regimen contemplated,pharmaceutical compositions of the present invention may be administeredparenterally, topically, orally, or locally. In certain aspects, thepharmaceutical compositions are administered parenterally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. In oneembodiment, the present invention provides compositions for parenteraladministration that comprise a compound of the present invention,dissolved or suspended in a carrier such as an aqueous carrier.

For solid formulations, compounds may be admixed with conventionalnontoxic solid carriers such as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like.

For aerosol administration, compounds of the present invention may besupplied in finely divided form along with a nontoxic surfactant andpropellant. Exemplary such agents are the esters or partial esters offatty acids containing from 6 to 22 carbon atoms, such as caproic,actanoic, lauric, palmitic, stearic, linoleic, olesteric, and oleicacids.

Compositions of the present invention may be administered by injection,i.e. intravenously, intramuscularly, intracutaneously, subcutaneously,introaduodenally, or intraperitoneally. Alternatively, compositions maybe administered by inhalation, such as intranasally, and may beadministered transdermally, such as through a patch or the like.

It will be understood that the actual preferred formulation ofcompositions, including pharmaceutical compositions, will vary accordingto the mode of administration as well as the particular disease beingtreated. The optimal formulations and modes of administration will beroutinely determined on a disease by disease and patient by patientbasis by those of skill in the art.

Methods for Identifying and Characterizing the In Vitro and In VivoEfficacy of Small-Molecule Modulators of Trp-p8

As discussed above, the present invention is directed to small-moleculeTrp-p8 modulators, including agonists and antagonists of Trp-p8activity. Disclosed herein are Trp-p8 modulators exemplified by thecompounds of Formula I, Formula II, Formula III, Formula IV, Formula V,Formula VI, Formula VII, and/or Formula VIII described herein above. Thepresent invention further contemplates that additional Trp-p8 modulatorsmay also be suitably employed in the compositions and methods of thepresent invention.

Additional or alternative Trp-p8 agonists and antagonists may beidentified by the methodology disclosed in the accompanying Examples.For instance, Trp-p8 agonists having efficacy in the treatment ofdisease(s) associated with Trp-p8 expression include small moleculesthat result in one or more of the following: (1) inhibit the growth ordecrease the viability of a cell expressing Trp-p8; (2) stimulatecalcium and/or other cation influx in a cell expressing Trp-p8; (3)induction of a poptosis and/or necrosis in a c ell expressing Trp-p8;and/or (4) efficacy in one or more animal model systems of humandisease. Trp-p8 antagonists having efficacy in the treatment ofdisease(s) associated with Trp-p8 expression include small moleculesthat that result in one or more of the following: (1) protect Trp-p8expressing cells from toxic effect of agonists in in vitro model system(2) inhibit growth of and/or kill cancer cell line with endogenousTrp-p8 expression (3) are efficacious in one or more animal modelsystems of human disease.

Thus, within certain embodiments, the present invention provides methodsfor identifying Trp-p8 agonists comprising the step of contacting aTrp-p8 expressing cell with a candidate Trp-p8 agonist for a time and inan amount sufficient to inhibit the growth and/or decrease the viabilityof a Trp-p8 expressing cell, wherein the inhibited growth and/or reducedviability indicate that the candidate Trp-p8 agonist is capable ofactivating Trp-p8 expressed by the cell.

Other embodiments provide methods for identifying Trp-p8 agonists,comprising the step of contacting a Trp-p8 expressing cell with acandidate Trp-p8 agonist for a time and in an amount sufficient toinduce influx of calcium and/or other cations into the cell, whereinincreased cation influx is correlative of increased cellular toxicity.

Still further embodiments provide methods for identifying Trp-p8agonists comprising the step of administering a candidate Trp-p8 agonistto an animal having one or more neoplastic cell that expresses Trp-p8for a time and in an amount sufficient to inhibit the growth of and/orinduce apoptosis and/or necrosis in the cell thereby increasing thesurvival of the animal, wherein any one or more of inhibition of cellgrowth, induction of apoptosis, induction of necrosis, and/or increasedsurvival of the animal indicate efficacy of the Trp-p8 agonist.

The present invention provides methods for the identification of Trp-p8antagonists in addition to the Trp-p8 antagonists disclosed herein bythe compounds of Formula VII and Formula VIII. Such method include (1)in vitro assay systems for detecting the protection of Trp-p8 expressingcells from toxicity induced by Trp-p8 agonists; (2) in vitro and in vivoassay systems of detecting the inhibition of growth of a cancer celland/or cancer cell line endogenously expressing Trp-p8; (3) in vivoanimal model systems whereby one or more candidate Trp-p8 antagonist isadministered to an animal having one or more neoplastic cell thatexpresses Trp-p8 for a time and in an amount sufficient to inhibit thegrowth of and/or induce apoptosis and/or necrosis in the cell therebyincreasing the survival of the animal.

Methods for Use of Trp-p8 Modulators

Small-molecule Trp-p8 modulators of the present invention may besuitably employed in methods for modifying (i.e. activating or reducing)Trp-p8-mediated calcium influx in a cell and therapeutic methods for thetreatment of one or more diseases associated with expression of Trp-p8.For example, and as noted above, it has been observed that abnormalTrp-p8 expression is associated with a neoplastic phenotype in a varietyof cancerous tissues including breast, colon, lung, and prostatetissues. Tsavaler et al., Cancer Research, supra.

Thus, within certain embodiments are provided methods for activatingTrp-p8-mediated calcium influx in a cell, such methods comprising thestep of contacting the Trp-p8 expressing cell with an amount of a Trp-p8agonist for a time sufficient to inhibit growth of the cell and/or toinduce necrosis and/or apoptosis in the cell. Exemplary methods foractivating Trp-p8 are provided within the Examples presented herein.

Other embodiments of the present invention provide therapeutic methodsfor the treatment of diseases associated with expression of Trp-p8, suchmethods comprising the step of administering to a mammal, typically ahuman, a therapeutically effective amount of a composition comprising aTrp-p8 agonist for a time sufficient to inhibit growth of the celland/or to induce necrosis and/or apoptosis in the cell. As used herein,the phrase “therapeutically effective amount” refers to the amount of acompound that, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending upon thecompound, the disease, and its severity and the age, weight, etc., ofthe mammal to be treated.

As used herein, the terms “treat”, “treating”, and “treatment” include:(1) preventing the disease, i.e. causing the clinical symptoms of thedisease not to develop in a mammal that may be predisposed to thedisease but does not yet experience any symptoms of the disease; (2)inhibiting the disease, i.e. arresting or reducing the development ofthe disease or its clinical symptoms; or (3) relieving the disease, i.e.causing regression of the disease or its clinical symptoms.

While the frequency and dosage of treatment regimens will vary dependingupon such factors as the disease and patient treated, compositionscomprising one or more compound of Formula I, Formula II, Formula III,Formula IV, Formula V, Formula VI, Formula VII, and/or Formula VIII aretypically administered in the range of about 0.001 mg compound/kg bodymass to about 100 mg/kg. Typically, treatment is initiated with smallerdosages that are less than the optimum dose of the compound. Thereafter,the dosage may be increased until optimal effectiveness is achieved.

In most instances, administration of a composition(s) of the presentinvention is achieved by any method that ensures systemic exposure tothe compound of Formula I, Formula II, Formula III, Formula IV, FormulaV, Formula VI, Formula VII, and/or Formula VIII. Thus, compositions maybe administered orally, parenterally, intraduodenally, and intranasally.Typically, such compositions comprise one or more such compound incombination with one or more pharmaceutically acceptable carrier ordiluent, as described in further detail herein above.

Other embodiments of the present invention provide combination therapieswherein one or more compound of Formula I, Formula II, Formula III,Formula IV, Formula V, Formula VI, Formula VII, and/or Formula VIII isadministered in conjunction with one or more cancer therapeutic agent,as described in further detail herein above, such as an antimitotic agent, an alkylating agent, an antimetabolite, a cytotoxic antibiotic, aplatinum agent, a hormonal agent, and/or an antiandrogen. Still furtherembodiments of the present invention provide combination therapieswherein two or more compounds of Formula I, Formula II, Formula III,Formula IV, Formula V, Formula VI, Formula VII, and/or Formula VIII areadministered either simultaneously or sequentially to achieve thedesired therapeutic outcome.

Thus, as used herein, the term “combination” means that at least twocompounds can be delivered in a simultaneous manner, in combinationtherapy wherein the first compound is administered first, followed bythe second compound, as well as wherein the second compound is deliveredfirst, followed by the first compound. The desired result can be eithera subjective relief of a symptom(s) or an objectively identifiableimprovement in the recipient of the dosages.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Synthesis of Menthane-3-carboxamide Compounds

This example discloses methodology for the synthesis ofmenthane-3-carboxamide Trp-p8 modulators.

Menthane-3-carboxylic acid (2).

Water (300 ml) was placed in a 2-L Erlenmeyer flask with a large stirbar. Sulfuric acid (500 ml) was added carefully with stirring. Thesolution was allowed to cool to 75° C., andN-ethyl-p-menthane-3-carboxamide (1, 62.5 g) was added. The temperaturewas maintained at 75° C. with a hot plate, and sodium nitrite (31 g) wasadded carefully. Two more 31 gram portions of NaNO₂ were added at 1-hourintervals, and the mixture was stirred overnight at 75° C.

The mixture was cooled to room temperature, diluted with ˜1 L of icewater, and extracted with ˜500 ml of ether. The ether layer wasseparated, washed with water, and extracted with 2×350 ml of 1M NaOH.The aqueous layer was made acidic with concentrated HCl and extractedwith ether. The ether layer was dried with MgSO₄ and evaporated to givementhane-3-carboxylic acid (33.2 g, 61%) as a crystalline solid,[□]=−50.3 deg (c=1, CHCl₃, 25° C.).

Menthane-3-carbonyl chloride (3).

Menthane-3-carboxylic acid (54.35 g) was refluxed with 80 ml of thionylchloride for 3 hours. The SOCl₂ was removed by distillation, and theacid chloride was distilled at 114-115° C. (8 Torr). (Lit. b.p. 84-85°C. at 3.5 Torr). Yield: 50 g (84%).

General Procedure for Preparation of menthane-3-carboxamides (5).

To a stirred solution of 0.2 mmol of the amine (4) in 1 ml ofacetonitrile or NMP and 0.4 mmol of DIPEA was added 0.022 ml ofmenthane-3-carbonyl chloride (3). The reaction mixture was shaken for 3hours. For less reactive amines, the mixture was heated (60° C.) andshaken for 24 hours. The product was purified from the crude reactionmixture by HPLC (40-95% gradient over 10 minutes using 0.05% TFA inCH₃CN and 0.05% TFA in H₂O) and evaporated to dryness.

Example 2 Synthesis of Menthol Acetamide and Carbamate Compounds

This example discloses methodology for the synthesis of MentholAcetamide and Carbamate Trp-p8 modulators.

General Procedures for the Preparation of Menthol Acetamides (7) andMenthol Carbamates (9).

To a two-phase ether-water system containing 0.07 mmol of amine (4) in 1ml of ether and 1 ml of 0.1 M aqueous sodium hydroxide was added 0.1mmol of acid chloride (6) or chloroformate (8). The reaction mixture wasshaken for 2-3 hours. The upper layer was removed and evaporated todryness to afford the product.

Example 3 Synthesis of Menthyl-3-urea and Menthyl-3-thiourea Compounds

This example discloses methodology for the synthesis of Menthyl-3-ureaand Menthyl-3-thiourea Trp-p8 modulators.

Menthyl-3-isocyanate (10).

To an ice-cooled solution of menthane-3-carboxylic acid (4 g, 22 mmol)and triethylamine (3.05 ml, 22 mmol) in dry DMF (45 ml) was addeddiphenylphosphoryl azide (4.72 ml, 22 mmol). The mixture was stirred at0° C. for 2 hours and at room temperature for 3 hours, then poured intoa mixture of ether and ice water. The ether layer was separated andwashed with aqueous sodium bicarbonate, dried with MgSO₄, andevaporated. The residue was distilled (85° C., 10 Torr) to givementhyl-3-isocyanate (2.9 g, 73%) as a colorless liquid.

General Procedure for the Preparation of menthyl-3-ureas (12) andmenthyl-3-thioureas (13).

To a stirred solution of 0.07 mmol of the amine (4) or thiol (11) in 1ml of dry ethyl acetate was added a solution of 0.07 mmol ofmenthyl-3-isocyanate in 0.5 ml of ethyl acetate. The reaction mixturewas shaken overnight then evaporated to dryness to afford the product.

Example 4 Synthesis of Menthane-3-amide, Menthane-3-sulfonamide andMenthane-3-carbamate Compounds

This example discloses methodology for the synthesis ofMenthane-3-amide, Menthane-3-sulfonamide and Menthane-3-carbamate Trp-p8modulators.

Menthyl-3-amine hydrochloride (14).

To a vigorously stirred mixture of diethyl ether (100 ml) and 6N aqueoushydrochloric acid (100 ml) was added menthyl-3-isocyanate (2.0 g, 11mmol), and the mixture was stirred overnight at room temperature. Theaqueous phase was separated, made basic with an excess of aqueous sodiumhydroxide, and extracted with ether. The ether phase was dried withMgSO₄ and filtered. An excess of anhydrous HCl (3M) in ethanol wasadded, and a precipitate immediately formed. Filtration gavementhylamine hydrochloride (1.9 g, 90%) as fine white crystals.

General Procedure for the Preparation of menthane-3-amides (18),menthane-3-sulfonamides (19) and menthane-3-carbamates (20).

A solution of menthyl-3-amine hydrochloride (0.078 mmol) in 0.7 ml ofwater was added to a solution of the acid chloride, sulfonyl chloride,or chloroformate (0.078 mop in 1 ml Et₂O, followed by the addition 0.3ml of 0.5 M NaOH. The mixture was shaken at room temperature for 12-18hours. The Et₂O layer was then separated and the solvent removed underreduced pressure to afford the product.

Example 5 Synthesis of Menthyl-3-amine Compounds

This example discloses methodology for the synthesis of Menthyl-3-amineTrp-p8 modulators.

General Procedures for the Preparation of menthyl-3-amines (23).

Method 1

A mixture of the amine (22, 0.1 mmol) and 15.4 mg (0.1 mmol) of menthone(21) in 1 ml of 1,2-dichloroethane was treated with sodiumtriacetoxyborohydride (32 mg, 0.15 mmol). The mixture was stirred for 24h (periodically monitored by LCMS). The reaction mixture was quenched byadding 1N NaOH (1 ml) and the product was extracted with ether. Theextract was washed with brine, dried (MgSO₄) and evaporated to drynessto afforf the product.

Method 2

A mixture of menthyl-3-amine (0.1 mmol) and 15.4 mg (0.1 mmol) ofaldehyde (21) in 1 ml of 1,2-dichloroethane was treated with sodiumtriacetoxyborohydride (32 mg, 0.15 mmol). The mixture was stirred for 24h (periodically monitored by LCMS). The reaction mixture was quenched byadding 1N NaOH (1 ml) and the product was extracted with ether. Theextract was washed with brine, dried (MgSO₄) and evaporated to dryness.

Example 6 Expression of Trp-p8 in CHO Cells

Human trp-p8 transfected CHO cells (referred to herein as CHO/Trp-p8)were generated for use in experiments of the present invention.Expression of Trp-p8 polypeptide in this transfectant and the absence ofany endogenous expression in the non-transfected to CHO was confirmed bywestern blot and immunofluorescence using a Trp-p8 specific antibody(GS2.20; disclosed within copending U.S. patent application Ser. No.10/______ incorporated herein by reference in its entirety) as well asthe calcium flux assay with Icilin(1-[2-hydroxyphenyl]-4-[3-nitrophenyl]-1,2,3,6-tetrahydropyrimidine-2-one)and menthol (2-isopropyl-5-methyl-cyclohexanol). Non-transfected CHOcells were used to establish the specificity of the effects of thecompounds observed with CHO/Trp-p8.

Example 7 Trp-p8-Mediated Decrease in Cell Viability Following Exposureof CHO/Trp-p8 Cells with Candidate Trp-P8 Agonist Compounds at 37° C.

This Example discloses an ATP viability assay suitable for screening foreffective Trp-p8 agonists. The ATP viability assay described hereinemploys CHO cells expressing an exogenous Trp-p8 cDNA. This examplefurther establishes that Trp-p8 agonists of the present invention areeffective in decreasing the viability of Trp-p8 expressing cells.

The concentration of intracellular ATP declines very rapidly whenmetabolically active cells undergo necrosis and/or apoptosis. The ATPconcentration and consequently the relative cell viability can bemeasured by established methods using commercially available reagents.In the agonist screening methodology disclosed herein, a compound thatspecifically decreases the viability of CHO/Trp-p8 cells is referred toas an agonist.

As a primary screen for efficacy and specificity for agonists, both thenon-transfected CHO and CHO/Trp-p8 cells were exposed to 10 μM of testcompounds in 1% dimethylsulfoxide (DMSO) or 1% DMSO (control) in a96-well black walled, black-bottomed, cell-culture treated plate. DMSOwas the solvent for all of the compounds tested. After 24-26 hours at37° C., the cells were lysed and ATP concentration determined via achemiluminescence assay using a commercially available reagent kit—CellTiter-Glo (Promega; Madison, Wis.). Relative viability (%), expressed asthe ATP level in cells treated with compounds expressed as a percentageof ATP levels in cells treated with the DMSO alone, was a measure of theagonist activity of the candidate compound—the lower the % viability,the more potent the Trp-p8 agonist. EC₅₀ values were determined for themost active candidate Trp-p8 agonists at 37° C. by measuring viabilityat 8-10 agonist concentrations. (EC₅₀ is defined herein as the agonistconcentration at which there is a 50% reduction in cell viability).

Exemplary Trp-p8 Agonists of Formula IV that were efficacious in the ATPviability assay are presented herein in Table 1. Table 1A presentsFormula IV Trp-p8 agonists exhibiting an EC₅₀ value within the range of0.05 to 0.20 μM; Table 1B presents Formula IV Trp-p8 agonists exhibitingan EC₅₀ value within the range of 0.20 to 0.50 μM; Table 1C presentsFormula IV Trp-p8 agonists exhibiting an EC₅₀ value within the range of0.50 to 1.00 μM; and Table 1D presents Formula IV Trp-p8 agonistsexhibiting an EC₅₀ value within the range of 1.00 to 7.00 μM.

Viability of CHO/Trp-p8 cells following treatment with exemplary Trp-p8agonists is presented in FIG. 1.

TABLE 1A Exemplary Trp-p8 Agonists of Formula IV Exhibiting an EC₅₀Value within the Range of 0.05 to 0.20 μM Ref. No.: Chemical StructureR¹⁴/R¹⁵ R¹⁵ 2905

2-hydroxy-2-phenylethyl 3012

2-oxo-2-phenylethyl 2896

2-hydroxy-2-(3- hydroxyphenyl)ethyl 3006

1-methyl-2-hydroxy-2- phenylethyl 2926

2-phenylethenyl 3014

Benzoylamino 2963

4-acetylphenyl

TABLE 1B Exemplary Trp-p8 Agonists of Formula IV Exhibiting an EC₅₀Value within the Range of 0.20 to 0.50 μM Ref. No.: Chemical StructureR¹⁴/R¹⁵ R¹⁵ 3024

N′-quinoxalin-2-yl-amino 2913

2-(4-hydroxyphenyl)ethyl 2897

2-hydroxy-2-(4- hydroxyphenyl)ethyl 2928

2-(3-hydroxyphenyl)ethyl 2901

Phenylcyclopropyl 1906

2-(2-furyl)ethyl 2920

2-(2-methylphenyl)ethyl 2952

2-(6-fluoro-1H-indol-3-yl)- ethyl 3013

2-(4-methoxy-phenyl)-2- oxo-ethyl 1603

2-phenylethyl 2264

2-(2-flourophenyl)ethyl 2261

2-hydroxy-2-phenylethyl 2904

2-hydroxy-2-phenylethyl 2932

2-(2,4-dichlorophenyl)ethyl 2931

2-(2-chloro-6- flourophenyl)ethyl 2942

2-(3-methylphenyl)ethyl 2930

2-(3-chlorophenyl)ethyl 1901

2-(2-methylphenyl)ethyl 2944

1-methyl-2-(5-fluoro-1H- indol-3-yl)-ethyl 3003

N-(2-diethylamino-ethyl)- benzamide-4-yl 2966

4-methylsulfanylphenyl 2973

2-chloro-4-cyanophenyl 2869

4-(2-hydroxyethyl)phenyl 2984

4-methyl-2-oxo-2H- chromen-7-yl 2832

4-(1-hydroxyethyl)phenyl 2836

3-oxo-indan-5-yl 2887

4-[2-(2-methoxy-phenyl)- 1,3-dioxo-2,3-dihydro-1H-isoindol-5-yloxyl-phenyl 2892

4-{2-[2-(3,4-dimethoxy- phenyl)-ethyl]-1,3-dioxo-2,3-dihydro-1H-isoindol-5- yloxy}-phenyl 2858

3-chloro-4-morpholin-4-yl- phenyl 2958

4-hydroxymethylphenyl 2864

2-chloro-4-iodophenyl 2831

4-carboxamidophenyl 2983

2-chloro-4-nitrophenyl 2961

3,4-cyclopentanephenyl 2828

4-cyanophenyl 2964

4-ethoxyphenyl

TABLE 1C Exemplary Trp-p8 Agonists of Formula IV Exhibiting an EC₅₀Value within the Range of 0.50 to 1.00 μM Ref. No.: Chemical StructureR¹⁴/R¹⁵ R¹⁵ 3040

2-(pyridin-3-yl)-ethyl 2903

2-methyl-2-phenylethyl 1903

2-thiophen-2-yl-ethyl) 2679

2-(1H-indol-3-yl)ethyl 2918

2-(3-methoxy-4- hydroxyphenyl)ethyl 2263

2-(4-flourophenyl)ethyl 3041

2-(pyridin-4-yl)-ethyl 3039

2-(pyridin-2-yl)-ethyl 1619

3-phenyl-piperidin-1-yl 2262

2-(3-flourophenyl)ethyl 2940

2-(2-methoxy-5- bromophenyl)ethyl 2270

1-hydroxymethyl-2-phenyl ethyl 3009

2-methyl-2-phenylethyl 2939

2-(3-bromo-4- methoxyphenyl) ethyl 2914

2-(4-methylphenyl) ethyl 3010

1-oxo-2-phenylethyl 2912

2-(4-bromophenyl)-ethyl 2922

2-phenyl-2-(4- flourophenyl)-ethyl 2950

2-(6-methoxy-1H-indol-3- yl)-ethyl 2868

4-methylphenyl 2891

4-[1,3-dioxo-2-(2- trifluoromethyl-phenyl)- 2,3-dihydro-1H-isoindol-5-yloxy]-phenyl 2998

4-(4-methyl-piperazin-1- ylmethyl)-phenyl 2960

C-1H-Indazol-5-yl 2970

2-flouro-4-chlorophenyl 2979

4-triflouromethylphenyl 2993

2-methyl-4-broophenyl 2987

4-Pyrrolidin-1-ylmethyl- phenyl 2853

2-phenyl-1H- benzoimidazol-5-yl 2875

4-(morpholine-4-sulfonyl)- phenyl 2956

2,4-dimethylphenyl 2978

3-chloro-4-methoxyphenyl 2856

2-pyridin-3-yl-1H- benzoimidazol-5-yl

TABLE 1D Exemplary Trp-p8 Agonists of Formula IV Exhibiting an EC₅₀Value within the Range of 1.00 to 7.00 μM Ref. No.: Chemical StructureR¹⁴/R¹⁵ R¹⁵ 2943

2-(2,5-dimethylphenyl)- ethyl 2917

2-(3-hydroxy-4- methoxyphenyl)-ethyl 2269

2-(2-methoxyphenyl)-ethyl 3007

1-methoxymethyl-2- phenylethyl 2898

1-methyl-2-(4- chlorophenyl)-ethyl 1627

3-phenyl-pyrrolidin-1-yl 2271

2-(2-chlorophenyl)ethyl 2933

2-(2,6- dimethylphenyl)ethyl 2936

2-(3,4-dichlorophenyl)ethyl 2923

2-phenyl-2-(4- methoxyphenyl)ethyl 2919

2-(2,4- dimethylphenyl)ethyl 2266

1-hydroxymethyl-2-phenyl ethyl 2929

2-(3- triflouromethylphenyl)ethyl 2935

2-(2,5- dimethoxyphenyl)ethyl 1591

2-cyclohex-1-enyl-ethyl 3035

benzyloxy 1568

2-(4-chlorophenyl)ethyl 2894

1-hydroxymethyl-2-(4- chlorophenyl)ethyl 2265

2-(4-methoxyphenyl)ethyl 2924

2-phenyl-2-(4- chlorophenyl)ethyl 2677

3-phenylpropyl 1910

2-(4- methylsulfanylphenyl)ethyl 2273

1-hydroxymethyl-2- hydroxy-2-phenylethyl 2937

2-(3,5- dimethoxyphenyl)ethyl 2949

2-(5-methoxy-1H-indol-3- yl)-ethyl 2941

2-(3-ethoxyphenyl)ethyl 2953

2-(7-methyl-1H-indol-3- yl)-ethyl 2938

2-(4-ethylphenyl)ethyl 2934

2-(2,3- dimethoxyphenyl)ethyl 2268

1-methyl-2-hydroxy-2- phenylethyl 2647

6,7-dimethoxy-1-methyl- 3,4-dihydro-1H- isoquinolin-2-yl 2915

2-(4-sulfamoyl-phenyl)- ethyl 1912

2-(1-cyclopentyl- pyrrolidin-3-yl)-ethyl 3032

2-(3-methoxy-4- ethoxyphenyl)ethyl 2947

1-hydroxymethyl-2-(1H- indol-3-yl)ethyl 2945

[1-carbamoyl-2-(1H-indol- 3-yl)-ethyl 1599

1-phenyl- cyclopentylmethyl 3008

1-hydroxymethyl-2-phenyl ethyl 2909

1-hydroxymethyl-2-(4- hydroxyphenyl)ethyl 2598

4-nitrobenzyl 2593

2,3-diflourobenzyl 2899

1-carbamoyl-2-phenylethyl 2279

2,2-diphenylethyl 2267

2-(3-methoxyphenyl)ethyl 1611

4-pyrimidin-2-yl- piperazin-1-yl 2277

2-(3,4- dimethoxyphenyl)ethyl 1566

4-methylcyclohexyl 2260

indan-2-yl 2900

1-carbamoyl-2-(4- hydroxyphenyl)ethyl 1637

cycloheptyl 1629

bicyclo[2.2.1]hept-2-yl 1614

2-(N,N- dipropylamino)ethyl 2272

2-(4-nitrophenyl)ethyl 2981

4-Biphenyl 1617

3,4-dimethylphenyl 2862

4-(1,1-dioxo-116- thiomorpholin-4-ylmethyl)- phenyl 2844

2-bromo-4-methylphenyl 2870

2-oxo-2,3-dihydro-1H- benzoimidazol-5-yl 3030

2-triflouromethyl-1H- benzoimidazol-5-yl 2974

2,3-dimethoxyphenyl 2849

4-Azepan-1-ylmethyl- phenyl 2850

4-(4-ethyl-piperazin-1-yl)- phenyl 1631

4-chlorophenyl 2841

1,3-dioxo-2,3-dihydro-1H- isoindol-4-yl 2843

3-bromo-4-methylphenyl 1607

4-methoxyphenyl 2840

2,4-dichlorophenyl 2962

3-yl-acetophenone 2872

2-flouro-5-nitrophenyl 2985

2-methyl-1,3-dioxo-2,3- dihydro-1H-isoindol-5-yl 1586

3-chloro-4-methylphenyl 2833

3-nitrophenyl 2957

3-hydroxymethylphenyl 1639

4-bromophenyl 2835

2-chloro-4-flourophenyl 2982

2-chloro-5-nitrophenyl 2954

2-methylphenyl 2871

3-acetamidophenyl 1575

4-ethylphenyl 2980

3,4-dichlorophenyl 1642

benzo[1,3]dioxol-5-yl 2873

1,3-dioxo-2,3-dihydro-1H- isoindol-5-yl 2965

2-nitrophenyl 2976

2-nitro-4-flourophenyl 1545

2,4-diflourophenyl 2990

3,4,5-trimethoxyphenyl 2829

2-chlorophenyl 2837

2-methyl-5-nitrophenyl 2859

2-iodophenyl 2972

3,4,5-triflourophenyl 3002

4-(4-methyl-1H- benzoimidazol-2-yl)-phenyl 2851

4-benzooxazol-2-yl-phenyl 1616

4-flourophenyl 2855

2-pyridin-2-yl- benzooxazol-5-yl 2830

3-carboxamidophenyl 1577

2,4-dimethoxyphenyl 1585

3,5-dimethoxyphenyl

Example 8 Screen and Characterization of Trp-p8 Agonist Compounds byMeasuring Calcium Influx in CHO/Trp-p8 Cells at 37° C.

This example discloses a CHO/Trp-p8-based calcium influx assay used tofurther assess the activity of candidate Trp-p8 agonists of the presentinvention.

Calcium influx was measured using a Flexstation Microplate FluorescencePlate Reader (Molecular Devices; Sunnyvale, Calif.). A typical assay forcalcium flux was performed as follows. Cells in DMEM/Ham's F-12 basedmedium, typically at a density of 30,000 cells/well/100 μl, were platedin a 96-well black-walled, clear bottomed tissue culture plate (GreinerBio-one) and incubated for 16-20 hours at 37° C. Cells in each well wereincubated for one hour at 37° C. with a Fura2-AM FluorescentDye/Pluronic F-27 mixture (Molecular Probes; Eugene, Oreg.) anddissolved in the medium containing Probenecid. Typical finalconcentrations were: 5-8 μM of Fura2-AM, 0.01% Pluronic F-27, and 2.5 mMProbenecid (an anion exchange inhibitor that reduces transport of thehydrolyzed dye from inside the cell thereby minimizing loss of dyeduring the experiment). After one hour, cells were washed in a bufferedsolution (20 mM HEPES and Hanks Balanced Salt Solution with 1.26 mMCaCl₂), pH 7.4 containing Probenecid at a final concentration of 2.5 mMand pre-incubated for at least 30 minutes at the assay temperature of37° C.

Typically, the above described HEPES/HBSS-based buffer containing eitherno additional calcium or with calcium to increase the concentration to 2mM and various concentrations of compounds (at 5-times the finalconcentrations) were added to each well using the robotic multi-channelpipettor. The compounds were preincubated at 37° C. for at least 30minutes before performing the assay (at 37° C.). Signals were read withdual excitation wavelengths of 340 and 380 nm and emission wavelength of510 nm with a cut-off filter at 495 nm. The signal was reported as theratio of emission when excited at 340 nm to the emission when excited at380 nm [Relative Fluorescence Units (RFU)]. Ionomycin was routinely usedas a positive control.

In the case of the agonist assay, the compounds at differentconcentrations were added to the dye-loaded cells (as described above).The increase in RFU was a measure of potency of the compound as anagonist. Exemplary results are presented in FIG. 2.

Example 9 Increase in Apoptosis Following Exposure of CHO/Trp-p8 Cellswith Trp-P8 Agonist Compounds at 37° C.

This example discloses the effectiveness of Trp-p8 agonist compounds ininducing apoptosis in Trp-p8 expressing cells.

An Annexin V/Propidium Iodide (PI) flow cytometry assay was used toprovide additional insights into the mechanism of cell death induced byTrp-p8 agonist compounds. Annexin V staining detects translocation ofPhosphatidylserine to the outer layer of plasma membrane, an eventcharacteristic of apoptosis, while PI staining indicates dead cells withcompromised membranes.

Cells were treated with compounds in 1% DMSO or with a 1% DMSO (control)for 24-26 hours at 37° C. The cells were briefly trypsinized undercontrolled conditions and stained with an Annexin V/PI reagent kitfollowing the methodology provided by the supplier (e.g., SouthernBiotech; Birmingham, Ala.). Exemplary results are presented in FIG. 3.

Example 10 In Vitro Screen Using a Cell Viability Assay for Trp-p8Antagonist Compounds Based Upon Protection of Trp-p8-Expressing Cellsfrom Toxic Agonist Compounds

This example discloses an assay system for identifying andcharacterizing candidate Trp-p8 antagonist compounds.

Trp-p8 antagonists were identified by employing a cell viability assaywith CHO/Trp-p8 cells at 37° C. (see Example 7) with the followingmodification. Within the context of the present invention, compoundsthat protect CHO/Trp-p8 cells from the toxic effect of a control agonistthereby maintaining the viability of the CHO/Trp-p8 cell exposed to aTrp-p8 agonist is defined as antagonist. As a primary screen forantagonists, CHO/Trp-p8 cells were exposed to 10 μM of test compounds in1% dimethylsulfoxide (DMSO) or 1% DMSO plus a toxic concentration of acontrol agonist, D-1607. The relative viability at 10 μM, determined asdescribed in Example 7, was a measure of the potential of the compoundas a Trp-p8 antagonist—the higher the viability, the more potent theantagonist. Exemplary results are presented in FIG. 4.

TABLE 2 Exemplary Formula VIII Trp-p8 Antagonists of Formula IV AgonistCompounds Relative Viability Ref. (%) at No.: Chemical Structure R²³/R²⁴R²⁴ 10 μM 1457

Tetrahydro isoquinolinyl 112 1465

Tetrahydro quinolinyl  90 1475

3-methyl indolinyl 100 1504

indolinyl  90 1582

2-(N-methyl, N- Phenylethyl) amino ethyl 103 1588

3-methyl indolinyl  88 1664

1-phenyl ethyl 107 1669

2-chloro benzyl  86 1673

2-methoxybenzyl 112 1688

Tetrahydro isoquinolinyl  91 1691

3-methyl indolinyl  92 1696

Tetrahydro quinolinyl  82 1709

2-methoxyphenyl  95 1743

2-cyclohex-1-enyl ethyl  97 1745

(1-Phenyl-cyclopentyl)- methyl  88 1781

3-methyl indolinyl  91 1815

2-(tetrahydroquinolinyl)- ethyl  87 1819

Tetrahydro isoquinolinyl  86 1838

1-Propyl-1,2,3,4- tetrahydro- pyrrolo[1,2-a] pyrazine  88 1876

cyclohepttyl  86 1882

3- Cyclohexylsulfanylpropyl  85 1883

2-cyclohex-1-enyl ethyl  84 1885

2-(N-isopropyl, N- Phenylethyl) amino ethyl  97 1918

1-methyl-1,2,3,4- tetrahydro- pyrrolo[1,2-a] pyrazine  94 1920

 81 1923

116 1925

 94 1937

101 1940

1-methyl-1,2,3,4- tetrahydro- pyrrolo[1,2-a] pyrazine 110 1941

1-Propyl-1,2,3,4- tetrahydro- pyrrolo[1,2-a] pyrazine  98 1996

2-cyclopentylethyl  89 2013

2-Phenylcyclopropyl  95 2018

1-phenoxyethyl 103 2044

4-butyloxyphenyl  94 2045

(2-nitrophenoxy)methyl 191 2046

4,7,7-trimethyl-2-oxa- bicyclo[2.2.1]heptan-3- one  89 2067

C-(1-Phenyl-5-propyl- 1H-pyrazol-4-yl)-methyl 105 2291

Benzyl  87 2306

2-chlorobenzyl  92 2639

benzyl; R2 = 2-(4- methyl)pyridyl 102 2676

1-[3-(6,7-Dimethoxy- 1-methyl-3,4- dihydro-1H- isoquinolin-2-ylmethyl)-4-methoxy- phenyl]-2,3,4,9- tetrahydro-1H-b- carboline  832777

C-[3-(4-Butoxy-phenyl)- 1H-pyrazol-4-yl]-methyl  83 2865

4-(Azepane-1-sulfonyl)- phenyl  91 3026

5-(7-Chloro-quinolin-4- ylsulfanyl)- [1,3,4]thiadiazol-2-yl  86 2131

2,4,6-trichlorophenyl  59 2134

4,5-dibromothiophen-2-yl  63 2710

2-hydroxy-5- methylphenyl  70 2745

3-phenyl-1H-pyrazol-4-yl  62 2752

3-(4-flourophenyl)-1H- pyrazol-4-yl  65 2754

3-(4-ethylphenyl)-1H- pyrazol-4-yl  74

TABLE 3 Exemplary Formula VII Trp-p8 Antagonists of Formula II AgonistCompounds Rel. Viab Ref. R¹⁹/ (%) at No. Chemical Structure R¹⁷ R¹⁸ R²⁰R²¹ 10 μM  13

2-Pyridyl 1-Benzyl-1H- pyrazolo[3,4-d] pyrimidin-4-yl H  91  27

2-Nitro-4- triflourome thylphenyl 3-benzylamino-2- nitrophenyl H  87  34

2-nitro-4- chlorophenyl 5-Nitro-quinolin- 8-yl H  82  36

2- methoxyphenyl 1-yl-3-(2- isopropyl-5- methyl- cyclohexyloxy)-propan-2-ol H  87  51

2- chlorophenyl 1-Phenyl-1H- pyrazolo[3,4-d] pyrimidin-4-yl H 100  67

phenyl Benzyl-2-methyl- quinazolin-4-yl H 102  69

phenyl 3-Methyl-5- morpholin-4-yl-2- nitro-phenyl H 106  74

2-methyl- quinolin-3- yl 2-nitro-5- piperazin-1-yl- ethanol H  81  93

4- methoxyphenyl 1-yl-3-(2- isopropyl-5- methyl- cyclohexyloxy)-propan-2-ol H 111  103

phenyl 4-(2,5-Dimethyl- pyrrol-1-yl)-2- nitro-phenyl H  99  107

phenyl 2-nitro-3- trifluoromethane- sulfonyl-phenyl H  98  159

4- flourphenyl 1-Phenyl-1H- pyrazolo[3,4-d] pyrimidin-4-yl H  95  711

phenyl 2-(2-Fluoro- phenoxymethyl)- 2-cyano oxazolyl H  94  809

3-azepan 1-yl-5-(4- triflouro- methoxy) phenylamino [1,3,5]triazyladamantyl H  89  812

3-azepan 1-yl-5-(4- triflouro- methoxy) phenylamino [1,3,5]triazyladamantyl H  99  881

cyclohexyl 5- (Benzo[1,3]dioxol- 5-ylamino)- 10b,10c-dihydro-anthra[1,9-cd] isoxazol-6- one-yl H  82  882

cyclohexyl 5- (Benzo[1,3]dioxol- 5-ylamino)- 10b,10c-dihydro-anthra[1,9-cd] isoxazol-6- one-yl H  86 1019

diphenylmethyl 2-Methyl- thiazolo[3,2-b] [1,2,4]triazol-6- ol4-methylphenyl methyl H  87 1021

diphenylmethyl 2-Methyl- thiazolo[3,2-b] [1,2,4]triazol-6- ol4-methylphenyl methyl H  92 1026

diphenylmethyl 2-Methyl- thiazolo[3,2-b] [1,2,4]triazol-6- ol4-methylphenyl methyl H  92 1027

diphenylmethyl 2-Methyl- thiazolo[3,2-b] [1,2,4]triazol-6- ol4-methylphenyl methyl H  84 1028

diphenylmethyl 2-Methyl- thiazolo[3,2-b] [1,2,4]triazol-6- ol4-methylphenyl methyl H  85 1039

diphenylmethyl 2-Methyl- thiazolo[3,2-b] [1,2,4]triazol-6- ol4-methylphenyl methyl H  84 1069

2- phenylethyl 3-benzyl-3H- quinazolin-4-one- 2-yl O  86 1262

4-hydroxy- cyclohexyl cyclopentyl O 4-methylphenyl  86 1280

cycloheptyl tetrahydronapthyl O 2-chloro-4- flourophenyl  87 1283

cyclopentyl cyclooctyl O 4-chlorophenyl 100 1284

cyclopentyl cyclohexyl O 4-chlorophenyl  95 1313

C- Benzo[1,3] dioxol-5- yl-methyl C-[3-(4-Chloro- phenyl)-2,5- dimethyl-pyrazolo[1,5-a] pyrimidin-7-yl]- methyl H  92 1323

2-Pyridyl C-(2-Benzyl- 5,6,7,8- tetrahydro- benzo[4,5]thieno[2,3-d]pyrimidin-4- yl)-methyl H  88 2496

1- chlorobenzyl 1-yl-3-(2- isopropyl-5- methyl- cyclohexyloxy)-propan-2-ol H  82

Example 11 In Vitro Screen Using a Calcium Flux Assay for Trp-p8Antagonist Compounds Based Upon their Abilities to Suppress the CalciumInflux Induced by Trp p8 Agonists in CHO/Trp-p8 Cells

This example discloses an in vitro assay system employed to furtherscreen and characterize candidate Trp-p8 antagonists.

Trp-p8 antagonists were also screened and characterized using a calciumflux assay at 37° C. as described in Example 8 with the following twodistinctions: (1) the compound was pre-mixed with the control agonist oronly the control agonist is added to the cells and suppression of theresponse to the agonist is a measure of the potency of the compound asan antagonist and (2) the compound, at different concentrations, wasadded to the cells followed by addition of the control agonist after 2-3minutes and the suppression of response induced by agonist was a measureof potency of the compound as an antagonist. Exemplary results arepresented in FIG. 5.

Example 12 An Animal Model System for Assaying the In Vivo Efficacy ofCandidate Trp-p8 Agonists and Antagonists for the Treatment of Cancer

This Example provides an animal model system suitable for determiningthe in vivo efficacy of candidate Trp-p8 modulators—including bothagonists and antagonists.

Human prostate cancer xenografts expressing Trp-P8 (from Dr. RobertVessella's lab in University of Washington—as assessed by in situhybridization and immunohistochemistry using a protein specific rabbitpolyclonal antibody, T-904) as well as CHO (Chinese Hamster Ovary) andEL-4 (Mouse Thymoma) cell lines were engineered to express Trp-P8 andused to establish tumor models in mice. Trp-P8 expression in thesetransfectants was confirmed by western blots and immunofluorescenceusing a Trp-p8 specific antibody (GS 2.20) as well as by response toknown agonists in a calcium influx functional assay. In addition, thesetransfected cell lines were susceptible to killing by Trp-p8 agonists asevident from the ATP viability and apoptosis assays (as described hereinin Examples 7 and 8).

A tumor model in mice was established by subcutaneously injectingCHO/Trp-P8 cells in SCID mice. Trp-p8 expression in tumors excised fromthese mice was confirmed by RT-PCR and western blot analysis. Furthertumor model development is carried out using the human prostate cancerxenografts described above in athymic nude or SCID mice and using anEL4/Trp-p8 transfectant in normal mice. Prostate xenografts from othersources and other cell lines that may be engineered to express Trp-p8are also potential candidates for building more model systems.

Based on results from in vitro and in vivo evaluations, a set of trp-p8agonists will be chosen to determine efficacy in mice. The in vitroevaluations would include potency in cell killing assay, aqueoussolubility, plasma binding study and metabolic stability (potential fora compound to be metabolized by liver as determined by using hepatocytesand/or mouse microsomes). The in vivo evaluations would includepharmacokinetics and toxicity studies. The chosen compounds will beadministered to mice with Trp-p8 expressing tumors by different routes[oral, intravenous, intraperitoneal, subcutaneous, intramuscular]. Tumorreduction and survival of these mice will be evaluated at differentdosages of these compounds. The compound most effective in fightingtumor will be chosen for further investigations

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding,changes and modifications can be carried out without departing from thescope of the invention which is intended to be limited only by the scopeof the appended claims.

1. A compound of Formula VIII

wherein R²² is a linker moiety, which may be selected from the groupconsisting of oxyacetamide, urea, carbamate, thiourea, sulfonamide,amine; R²³ is selected from the group consisting of H, an aliphaticgroup of up to 25 carbons, and an aryl group of up to 10 carbonsselected from the group consisting of substituted phenyl, phenalkyl,substituted phenalkyl, naphthyl, substituted naphthyl, and pyridyl; andR²⁴ is selected from the group consisting of H, OH, and an aliphaticgroup containing up to 25 carbon atoms; and R²⁵ is selected from thegroup consisting of H. 2.-4. (canceled)
 5. A composition comprising acompound of claim 1 and a pharmaceutically acceptable excipient ordiluent. 6.-7. (canceled)
 8. A method for decreasing the viability of aTrp-p8 expressing cell, said method comprising the step of contactingsaid cell with a small-molecule Trp-p8 modulator in a concentration andfor a time required to decrease the viability of said cell.
 9. Themethod of claim 8 wherein said small-molecule Trp-p8 modulator isselected from the group consisting of a compound of Formula I, acompound of Formula II, a compound of Formula III, a compound of FormulaIV, a compound of Formula V, a compound of Formula VI, a compound ofFormula VII, and a compound of Formula VIII.
 10. The method of claim 9wherein said small-molecule Trp-p8 modulator is a compound of Formula I

wherein R¹ is selected from the group consisting of H, OH, CH₃,CH₃—CH—CH₃ (isopropyl), and CH₃—C═CH₂ (isopropenyl); R² is selected fromthe group consisting of H; R³ is selected from the group consisting ofO, OH, acetate, lactate, carboxamide, butanamide, sulphanamide, andpropanetriol; and R⁴ is selected from the group consisting of CH₃—CH—CH₃(isopropyl), isopropane-2-ol, and CH₃—C═CH₂ (isopropenyl).
 11. Themethod of claim 9 wherein said small-molecule Trp-p8 agonist is acompound of Formula II

wherein R⁵ is selected from the group consisting of H, OH, CH₃,CH₃—CH—CH₃ (isopropyl), and CH₃—C═CH₂ (isopropenyl); R⁶ is selected fromthe group consisting of N; R⁷ is selected from the group consisting of Oand N; R⁸ is selected from the group consisting of NH, O, and S; and R⁹is selected from the group consisting of NO₂.
 12. The method of claim 9wherein said small-molecule Trp-p8 modulator is a compound of FormulaIII

wherein R¹⁰ is selected from the group consisting of H and a C₁-C₅ alkylincluding, but not limited to, CH₃, C₂H₅, CH₃—CH—CH₃ (isopropyl) andCH₃—C═CH₂ (isopropenyl); R¹¹ is selected from the group consisting ofOH, carboxamide, butanamide, propanetriol, and CONR′R″, wherein R′ isselected from the group consisting of H, CH₃, C₂H₅, C₄H₈ (cyclobutyl),and C₄H₈O, and wherein R″ is selected from the group consisting ofC₂H₅O00H₂, C₂₁-1₅, CH₃—CH—CH₃ (isopropyl), HOCH₂C(CH₃)₂, HOCH₂CH₂, C₄H₉(tertbutyl), and C₄1-1₉ (secbutyl); R¹² is selected from the groupconsisting of H and a C1-C5 alkyl including, but not limited to, CH₃,CH₃—CH—CH₃ (isopropyl), CH₃—C═CH₂ (isopropenyl), C₄H₉ (secbutyl), C₄H₉(isobutyl), C₄H₉ (n-butyl), and C₅H₁₁ (isohexyl); and R¹³ is selectedfrom the group consisting of H and a C₁-C₅ alkyl including, but notlimited to, CH₃, C₂H₅, CH₃—CH—CH₃ (isopropyl), CH₃—C═CH₂ (isopropenyl),C₄H₉ (secbutyl), and C₄H₉ (isobutyl).
 13. The method of claim 9 whereinsaid small-molecule Trp-p8 modulator is a compound of Formula IV

wherein R¹⁴ is selected from the group consisting of H, an aliphaticgroup of up to 25 carbons, and an aryl group of up to 10 carbons and R¹⁵is selected from the group consisting of H, OH, and an aliphatic groupcontaining up to 25 carbon atoms. 14-17. (canceled)
 18. The method ofclaim 9 wherein said small-molecule Trp-p8 modulator is a compound ofFormula V

wherein R¹⁶ is selected from the group consisting of a C₂-C₆ alkylenegroup having at least one, but not more than three, hydroxyl groups; andR¹⁷ and R¹⁸, independently of one another, are selected from the groupconsisting of C₁-C₁₀-alkyl groups, C₅-C₇-cycloalkyl, C₆-C₁₂-aryl,wherein the total of the C atoms of R¹⁷ and R¹⁸ is not less than 3, orR¹⁷ and R¹⁸ together represent an alkylene group that, together with thecarbon atom that carries the groups R¹⁷ and R¹⁸, forms a 5-7-memberedring.
 19. (canceled)
 20. The method of claim 9 wherein saidsmall-molecule Trp-p8 modulator is a compound of Formula VII

wherein R¹⁷ is selected from the group consisting of 2-pyridyl,2-nitro-4-trifluoromethylphenyl, 2-nitro-4-chlorophenyl,2-methoxyphenyl, 2-chlorophenyl, phenyl, 2-methyl-quinolin-3-yl,4-methoxyphenyl, 4-fluorophenyl,3-azepan1-yl-5-(4-trifluoromethoxy)phenylamino[1,3,5]triazyl,cyclohexyl, diphenylmethyl, 2-phenylethyl, 4-hydroxy-cyclohexyl,cycloheptyl, cyclopentyl, C-benzo[1,3]dioxol-5-yl-methyl, 2-pyridyl, and4-chlorobenzyl; R¹⁸ is selected from the group consisting of1-benzyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl, 3-benzylamino-2-nitrophenyl,5-nitro-quinolin-8-yl,1-yl-3-(2-isopropyl-5-methylcyclohexyloxy)-propan-2-ol,1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,benzyl-2-methylquinazolin-4-yl,3-methyl-5-morpholin-4-yl-2-nitro-phenyl,2-nitro-5-piperazin-1-yl-ethanol,1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol,4-(2,5-dimethyl-pyrrol-1-yl)-2-nitro-phenyl,2-nitro-3-trifluoromethanesulfonyl-phenyl,1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,2-(2-Fluoro-phenoxymethyl)-2-cyano oxazolyl, adamantly,5-(benzo[1,3]dioxol-5-ylamino)-10b,10c-dihydro-anthra[1,9-cd]isoxazol-6-one-yl,2-methylthiazolo[3,2-b][1,2,4]triazol-6-01 4-methylphenyl methyl,3-benzyl-3H-quinazolin-4-one-2-yl, cyclopentyl, tetrahydronapthyl,cyclooctyl, cyclohexyl,C-[3-(4-chloro-phenyl)-2,5-dimethyl-pyrazolo[1,5-a]pyrimidin-7-yl]-methyl,C-(2-benzyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-methyl, and1-yl-3-(2-isopropyl-5-methyl cyclohexyloxy)-propan-2-ol; R¹⁹ and R²⁰ areeach independently selected from the group consisting of H and O; andR²¹ is selected from the group consisting of 4-methylphenyl,2-chloro-4-fluorophenyl, and 4-chlorophenyl.
 21. The method of claim 9wherein said small-molecule Trp-p8 modulator is a compound of FormulaVIII

wherein R²² is a linker moiety, which may be selected from the groupconsisting of oxyacetamide, urea, carbamate, thiourea, sulfonamide,amine, amide; R²³ is selected from the group consisting of H, tetrahydroisoquinolinyl, tetrahydro quinolinyl, 3-methyl indolinyl, indolinyl,2-(N-methyl, N-phenylethyl)amino ethyl, 3-methyl indolinyl, 1-phenylethyl, 2-chloro benzyl, 2-methoxybenzyl, 2-methoxyphenyl,2-cyclohex-1-enyl ethyl, (1-phenyl-cyclophenyl)-methyl,2-(tetrahydroquinolinyl)-ethyl,1-propyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, cycloheptyl,3-cyclohexylsulfanylpropyl, 2-cyclohex-1-enyl ethyl, 2-(N-isopropyl,N-phenylethyl)amino ethyl,1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, 2-cyclopentylethyl,2-phenylcyclopropyl, 1-phenoxyethyl, 4-butyloxyphenyl,(2-nitrophenoxy)methyl,4,7,7-trimethyl-2-oxa-bicyclo[2.2.1]heptan-3-one,C-(1-phenyl-5-propyl-4H-pyrazol-4-yl)-methyl, benzyl, 2-chlorobenzyl,1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-methoxy-phenyl]-2,3,4,9-tetrahydro-1H-b-carboline,C-[3-(4-butoxy-phenyl)-1H-pyrazol-4-yl]-methyl,4-(azepane-1-sulfonyl)-phenyl, and5-(7-chloro-quinolin-4-ylsulfanyl)-[1,3,4]thiadiazol-2-yl; R²⁴ isselected from the group consisting of H, tetrahydro isoquinolinyl,tetrahydro quinolinyl, 3-methyl indolinyl, indolinyl, 3-methylindolinyl, 1-propyl-1,2,3,4-pyrrolo[1,2-a]pyrazine,1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, and1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-methoxy-phenyl]-2,3,4,9tetrahydro-1H-b-carboline; and R²⁵ is selected from the group consistingof H.
 22. A method for inducing apoptosis and/or necrosis in a cellexpressing Trp-p8 or for treating a disease associate with Trp-p8expression, said method comprising the step of administering to saidcell a small-molecule Trp-p8 modulator selected from the groupconsisting of a compound of Formula I, a compound of Formula II, acompound of Formula III, a compound of Formula IV, a compound of FormulaV, a compound of Formula VI, a compound of Formula VII, and a compoundof Formula VIII.
 23. A method for treating a disease associated withTrp-p8 expression of claim 22, said method comprising the steps ofadministering to a mammal an efficacious amount of a compositioncomprising a compound having the formula:

in combination with a pharmaceutically acceptable carrier or diluent,wherein R¹ is selected from the group consisting of H, OH, CH₃,CH₃—CH—CH₃ (isopropyl), and CH₃—C═CH₂ (isopropenyl); R² is selected fromthe group consisting of OH, carboxamide, butanamide, and propanetriol;and R³ is selected from the group consisting of CH₃—CH—CH₃ (isopropyl),isopropane-2-ol, and CH₃—C═CH₂ (isopropenyl).
 24. A method for treatinga disease associated with Trp-p8 expression of claim 22, said methodcomprising the step of administering to a mammal an efficacious amountof a composition comprising a compound having the formula:

in combination with a pharmaceutically acceptable carrier or diluent,wherein R⁵ is selected from the group consisting of H, OH, CH₃,CH₃—CH—CH₃ (isopropyl), and CH₃—C═CH₂ (isopropenyl); R⁶ is selected fromthe group consisting of N; R⁷ is selected from the group consisting ofO, CH₂, and CH₃—C—CH₃ (isopropyl); R⁸ is selected from the groupconsisting of NH; and R⁹ is selected from the group consisting of N02.25. A method for treating a disease associated with Trp-p8 expression ofclaim 22, said method comprising the step of administering to a mammalan efficacious amount of a composition comprising a compound having theformula:

in combination with a pharmaceutically acceptable carrier or diluent,wherein R¹⁰ is selected from the group consisting of H, CH₃, CH₃—CH—CH₃(isopropyl), and CH₃—C═CH₂ (isopropenyl); R¹¹ is selected from the groupconsisting of OH, carboxamide, butanamide, and propanetriol; R¹² isselected from the group consisting of H, CH₃, CH₃—CH—CH₃ (isopropyl),and CH₃—C═CH₂ (isopropenyl); and R¹³ is selected from the groupconsisting of H, CH₃, CH₃—CH—CH₃ (isopropyl), and CH₃—C═CH₂(isopropenyl); and a pharmaceutically acceptable carrier or diluent. 26.A method for treating a disease associated with Trp-p8 expression ofclaim 22, said method comprising the step of administering to a mammalan efficacious amount of a composition comprising a compound having theformula:

in combination with a pharmaceutically acceptable carrier or diluent,wherein R¹⁴ is selected from the group consisting of H, an aliphaticgroup of up to 25 carbons, and an aryl group of up to 10 carbons and R¹⁵is selected from the group consisting of H, OH, and an aliphatic groupcontaining up to 25 carbon atoms. 27.-30. (canceled)
 31. A method fortreating a disease associated with Trp-p8 expression of claim 22, saidmethod comprising the step of administering to a mammal an efficaciousamount of a composition comprising a compound having the formula:

in combination with a pharmaceutically acceptable carrier or diluent,wherein R¹⁶ is selected from the group consisting of a C₂-C₆ alkylenegroup having at least one, but not more than three, hydroxyl groups; andR¹⁷ and R¹⁸, independently of one another, are selected from the groupconsisting of C₁-C₁₀-alkyl groups, C₅-C₇-cycloalkyl, C₆-C₁₂-aryl,wherein the total of the C atoms of R¹⁷ and R¹⁸ is not less than 3, orR¹⁷ and R¹⁸ together represent an alkylene group that, together with thecarbon atom that carries the groups R¹⁷ and R¹⁸, forms a 5-7-memberedring.
 32. (canceled)
 33. A method for treating a disease associated withTrp-p8 expression of claim 22, said method comprising the step ofadministering to a mammal an efficacious amount of a compositioncomprising a compound having the formula:

in combination with a pharmaceutically acceptable carrier or diluent.34. A method for treating a disease associated with Trp-p8 expression ofclaim 22, said method comprising the step of administering to a mammalan efficacious amount of a composition comprising a compound having theformula:

in combination with a pharmaceutically acceptable carrier or diluent,wherein R¹⁷ is selected from the group consisting of 2-pyridyl,2-nitro-4-trifluoromethylphenyl, 2-nitro-4-chlorophenyl,2-methoxyphenyl, 2-chlorophenyl, phenyl, 2-methyl-quinolin-3-yl,4-methoxyphenyl, 4-fluorophenyl,3-azepan1-yl-5-(4-trifluoromethoxy)phenylamino[1,3,5]triazyl,cyclohexyl, diphenylmethyl, 2-phenylethyl, 4-hydroxy-cyclohexyl,cycloheptyl, cyclopentyl, C-benzo[1,3]dioxol-5-yl-methyl, 2-pyridyl, and4-chlorobenzyl; R¹⁸ is selected from the group consisting of1-benzyl-1-pyrazolo[3,4-d]pyrimidin-4-yl, 3-benzylamino-2-nitrophenyl,5-nitro-quinolin-8-yl,1-yl-3-(2-isopropyl-5-methylcyclohexyloxy)-propan-2-ol,1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,benzyl-2-methylquinazolin-4-yl,3-methyl-5-morpholin-4-yl-2-nitro-phenyl,2-nitro-5-piperazin-1-yl-ethanol,1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol,4-(2,5-dimethyl-pyrrol-1-yl)-2-nitro-phenyl,2-nitro-3-trifluoromethanesulfonyl-phenyl,1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,2-(2-Fluoro-phenoxymethyl)-2-cyano oxazolyl, adamantly,5-(benzo[1,3]dioxol-5-ylamino)-10b,10c-dihydro-anthra[1,9-cd]isoxazol-6-one-yl,2-methylthiazolo[3,2-b][1,2,4]triazol-6-01 4-methyl-phenyl methyl,3-benzyl-3H-quinazolin-4-one-2-yl, cyclopentyl, tetrahydronapthyl,cyclooctyl, cyclohexyl,C-[3-(4-chloro-phenyl)-2,5-dimethyl-pyrazolo[1,5-a]pyrimidin-7-yl]-methyl,C-(2-benzyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-methyl, and1-yl-3-(2-isopropyl-5-methyl cyclohexyloxy)-propan-2-ol; R¹⁹ and R²⁰ areeach independently selected from the group consisting of H and O; andR²¹ is selected from the group consisting of 4-methylphenyl,2-chloro-4-fluorophenyl, and 4-chlorophenyl.
 35. A method for treating adisease associated with Trp-p8 expression of claim 22, said methodcomprising the step of administering to a mammal an efficacious amountof a composition comprising a compound having the formula:

in combination with a pharmaceutically acceptable carrier or diluent,wherein R²² is a linker moiety, which may be selected from the groupconsisting of oxyacetamide, urea, carbamate, thiourea, sulfonamide,amine, amide; R²³ is selected from the group consisting of H, tetrahydroisoquinolinyl, tetrahydro quinolinyl, 3-methyl indolinyl, indolinyl,2-(N-methyl, N-phenylethyl)amino ethyl, 3-methyl indolinyl, 1-phenylethyl, 2-chloro benzyl, 2-methoxybenzyl, 2-methoxyphenyl,2-cyclohex-1-enyl ethyl, (1-phenyl-cyclophenyl)-methyl,2-(tetrahydroquinolinyl)-ethyl,1-propyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, cycloheptyl,3-cyclohexylsulfanylpropyl, 2-cyclohex-1-enyl ethyl, 2-(N-isopropyl,N-phenylethyl)amino ethyl,1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, 2-cyclopentylethyl,2-phenylcyclopropyl, 1-phenoxyethyl, 4-butyloxyphenyl,(2-nitrophenoxy)methyl,4,7,7-trimethyl-2-oxa-bicyclo[2.2.1]heptan-3-one,C-(1-phenyl-5-propyl-1H-pyrazol-4-yl)-methyl, benzyl, 2-chlorobenzyl,1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-methoxy-phenyl]-2,3,4,9tetrahydro-1H-b-carboline,C-[3-(4-butoxy-phenyl)-1H-pyrazol-4-yl]-methyl,4-(azepane-1-sulfonyl)-phenyl, and5-(7-chloro-quinolin-4-ylsulfanyl)-[1,3,4]thiadiazol-2-yl; R²⁴ isselected from the group consisting of H, tetrahydro isoquinolinyl,tetrahydro quinolinyl, 3-methyl indolinyl, indolinyl, 3-methylindolinyl, 1-propyl-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine,1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, and1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-methoxy-phenyl]-2,3,4,9tetrahydro-1H-b-carboline; and R²⁵ is selected from the group consistingof H.
 36. A method for identifying a Trp-p8 agonist, said methodcomprising the step of contacting a Trp-p8 expressing cell and anon-Trp-p8 expressing cell with a candidate Trp-p8 agonist for a timeand in an amount sufficient to decrease the viability of said Trp-p8expressing cell but not said non-Trp-p8 expressing cell.
 37. A methodfor identifying a Trp-p8 antagonist, said method comprising the step ofcontacting a Trp-p8 expressing cell with a Trp-p8 agonist and with acandidate Trp-p8 antagonist for a time and in an amount sufficient forsaid agonist to decrease the viability of said T rp-p8 expressing cell,wherein a Trp-p8 antagonist is detected by an increase in the viabilityof said Trp-p8 expressing cell.